Recombinant halohydrin dehalogenase polypeptides

ABSTRACT

The present disclosure provides engineered halohydrin dehalogenase (HHDH) polypeptides having improved enzyme properties as compared to the wild-type HHDH enzyme HheC and other reference engineered HHDH polypeptides. Also provided are polynucleotides encoding the engineered HHDH enzymes, host cells capable of expressing the engineered HHDH enzymes, and methods of using the engineered HHDH enzymes to synthesize a variety of chiral compounds including chiral epoxides and chiral alcohols.

1. CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority of U.S. provisional application61/138,943, filed Dec. 18, 2008, which is hereby incorporated byreference herein.

2. REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The Sequence Listing concurrently submitted herewith under 37 C.F.R.§1.821 in a computer readable form (CRF) via EFS-Web as file nameCX2-027US1_ST25.txt is herein incorporated by reference. The electroniccopy of the Sequence Listing was created on Dec. 17, 2009, with a filesize of 517 Kbytes. This Sequence Listing is identical except for minorformatting corrections to file 376247-036USP1.txt created on Dec. 18,2008, with a file size of 508 Kbytes, which was incorporated byreference in the priority U.S. provisional application 61/138,943.

3. BACKGROUND

Enzymes belonging to the halohydrin dehalogenase (HHDH), also namedhalohydrin hydrogen-halide-lyase or halohydrin epoxidase, class (EC4.5.1) catalyze the interconversion of 1,2-halohydrins and thecorresponding 1,2-epoxides as shown below in Scheme 1.

U.S. Pat. No. 4,284,723 describes the use of a halohydrin epoxidase forthe production of propylene oxide. U.S. Pat. Nos. 5,166,061 and5,210,031 describe the use of this enzyme activity for the conversion of1,3-dichloropropanol (DCP) and epichlorohydrin (ECH) respectively to4-chloro-3-hydroxybutyronitrile (CHBN). HHDH enzymes from Agrobacteriumradiobacter and Corynebacterium have been characterized on a broad rangeof halogenated substrates (Van Hylckama Vlieg et al., J. Bacteriol.(2001) 183:5058-5066; Nakamura et al., Appl. Environ. Microbiol. (1994)60:1297-1301; Nagasawa et al., Appl. Microbiol. Biotechnol. (1992)36:478-482).

HHDH also catalyzes the ring opening of epoxides with nucleophiles otherthan chloride or bromide as shown in Scheme 2 (see e.g., Nakamura etal., Biochem. Biophys Res. Comm. (1991) 180:124-130; Nakamura et al.,Tetrahedron (1994) 50: 11821-11826; Lutje Spelberg et al., Org. Lett.(2001) 3:41-43; Lutje Spelberg et al., Tetrahedron Asymm. (2002)13:1083).

Nakamura et al. (Tetrahedron (1994) 50: 11821-11826) describe the use ofHHDH for the direct conversion of DCP to chloro-3-hydroxy-butyronitrile(CHBN) through epichlorohydrin (ECH) as the intermediate as shown belowin Scheme 3.

Some halohydrin dehalogenases have been characterized. For example, HHDHfrom A. radiobacter AD1 is a homotetramer of 28 kD subunits.Corynebacterium sp. N-1074 produces two HHDH enzymes, one of which,enzyme Ia, is composed of 28 kD subunits, while the other, enzyme Ib. iscomposed of related subunits of 35 and/or 32 kD. HHDH from some sourcesis easily inactivated under oxidizing conditions in a process that leadsto dissociation of the subunits, has a pH optimum from pH 8 to 9 and anoptimal temperature of 50° C. (Tang, Enz. Microbial Technol. (2002)30:251-258; Swanson, Curr. Opin. Biotechnol. (1999) 10:365-369). Theoptimal pH for HHDH catalyzed epoxide formation has been reported as 8.0to 9.0 and the optimal temperature in the range of from 45° C. to 55° C.(see e.g., Van Hylckama Vlieg et al., J. Bacteriol. (2001)183:5058-5066; Nakamura et al., Appl. Environ. Microbiol. (1994)60:1297-1301; Nagasawa et al., Appl. Microbiol. Biotechnol. (1992)36:478-482). The optimal pH for the reverse reaction, ring opening bychloride, has been reported for the two Corynebacterium sp. N-1074enzymes Ia and Ib and is pH 7.4 and pH 5, respectively. Site directedmutagenesis studies on the A. radiobacter AD1 HHDH indicated thatoxidative inactivation is due to disruption of the quaternary structureof the enzyme by oxidation of cysteine residues (Tang et al., Enz.Microbial Technol. (2002) 30:251-258).

Purified HHDH enzymes from different sources exhibit specific activitieson DCP ranging from 146 U/mg (Ib) to 2.75 U/mg (Ia) (Nakamura et al.,Appl. Environ. Microbiol. 1994 60:1297-1301; Nagasawa et al., Appl.Microbiol. Biotechnol. (1992) 36:478-482). The high activity of the Ibenzyme is accompanied by a high enantioselectivity to produce R-ECH fromDCP, while the Ia enzyme produces racemic ECH.

HHDH encoding genes have been identified in Agrobacterium radiobacterAD1 (hheC), Agrobacterium tumefaciens (halB), Corynebacterium sp (hheAencoding Ia and hheB encoding Ib), Arthrobacter sp. (hheA_(AD2)), andMycobacterium sp. GP1 (hheB_(Gp1)). All enzymes have been functionallyexpressed in E. coli.

It is highly desirable for commercial applications that an HHDH enzymeexhibits high volumetric productivity, that reactions run to completionin a relatively short period of time, with a high final productconcentration, with high enantioselectivity and regioselectivity,exhibits minimal inhibition by reaction components, and that generationof undesirable chemical side products is reduced. It is also desirableto have an HHDH enzyme capable of catalyzing ring opening or ringclosure on a range of substrates (Hasnaoui-Dijoux et al., 2008, Chem.Biochem. 9:1048-1051; Tang et al., 2003, Biochemistry 42:5378-5386;Lutje Spelberg et al., 2000, Org. Lett. 3(1):41-43; Lutje Spelberg etal., 1998, Tetrahedron Asymmetry 9:459-466; and Majeric-Elenkov et al.,Organic Letters 10 (12), pg 2417-2420, 2008).

4. SUMMARY

Provided in the present disclosure are engineered halohydrindehalogenase (HHDH) polypeptides having altered or improved enzymeproperties, including, among others, changes to substrate recognition,enantioselectivity, halide ion resistance, and regioselectivity. Thesechanges allow the engineered HHDHs to be used in biocatalyticapplications for preparing compounds not efficiently produced by otherHHDH polypeptides.

In some embodiments the disclosure provides an engineered halohydrindehalogenase (HHDH) polypeptide having an improved enzyme propertyrelative to one or both of the reference polypeptides of SEQ ID NO: 244or SEQ ID NO: 4, where the engineered HHDH comprising an amino acidsequence at least 80% identical to SEQ ID NO: 244 or SEQ ID NO: 4, andat least one residue difference as compared to SEQ ID NO:244 at aposition selected from: X34; X51; X81; X85; X110; X131; X167; X232;X242; X248, and X254. In certain embodiments the residue difference isselected from: X34 is G; X51 is P; X81 is E, F, M, W, or Y; X85 is V;X110 is S; X131 is A; X167 is H; X232 is S; X242 is A; X248 is V; andX254 is N.

In some embodiments the disclosure provides an engineered HHDH having animproved enzyme property relative to one or both of the referencepolypeptides of SEQ ID NO: 244 or SEQ ID NO: 4, wherein the engineeredHHDH comprises an amino acid sequence at least 80% identical to SEQ IDNO:244 and has and at least one residue difference as compared to SEQ IDNO:244 selected from: X12 is M, or S; X34 is G; X51 is P; X81 is E, F,M, W, or Y; X82 is C, G, K, L, M, N, or W; X83 is C or E; X84 is D; X85is V; X86 is A or H; X110 is S, X131 is A; X134 is C, F, I, K, L, M, orV; X139 is G, I, L, M, P, S, T, or V; X142 is C, M or N; X146 is P; X167is H; X175 is C, G, I, L, M, N, or V; X176 is F, M, Q, or T; X177 is L,S or T; X178 is T; X182 is S; X186 is H; X187 is G, I, or S; X201 is R;X232 is S, X242 is A; X243 is C or L; X245 is S; X246 is S; X247 is N;X248 is V; X249 is A, G, H, I, or S; X252 is C; and X254 is N.

In some embodiments the disclosure provides an engineered HHDH having animproved enzyme property relative to one or both of the referencepolypeptides of SEQ ID NO: 244 or SEQ ID NO: 4 comprises an amino acidsequence selected from the group consisting of SEQ ID NO: 2, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82,84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142,144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170,172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198,200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226,228, 230, 232, 234, 236, 238, 240, 242, 246, 248, 250, 252, 254, 256,258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284,286, 288, 290, 292, 294, 296, 298, 300, 302, and 304.

In some embodiments the engineered HHDH polypeptides of the disclosureare capable of converting a racemic epoxide substrate of formula (I)(see infra, e.g., styrene oxide) to an S-alcohol product of formula (II)with an increased enantiomeric excess of product as compared toreference polypeptide of SEQ ID NO: 4 or 244.

In some embodiments the engineered HHDH polypeptides of the disclosureare capable of an increased R-stereoselectivity in converting theepoxide of formula (I) (see infra, e.g., styrene oxide) to the R-alcoholproduct of formula (III) as compared to a reference polypeptide of SEQID NO: 4 or 244.

In some embodiments the engineered HHDH polypeptides of the disclosureare capable of increased α-regioselectivity or β-regioselectivity in theconversion of an epoxide substrate of formula (I) (see infra, e.g.,styrene oxide) to a product of formula (II) as compared to a referencepolypeptide of SEQ ID NO: 4 or 244.

In some embodiments the engineered HHDH polypeptides of the disclosureare capable of increased refractoriness to inhibition by a halide ion ascompared to a reference HHDH polypeptide of SEQ ID NO: 4 or 244. In someembodiments, the halide ion is selected from Br⁻, Cl⁻, or I⁻.

In some embodiments the engineered HHDH polypeptides of the disclosureare capable of an increased activity for converting an epoxide substrateof formula (I) to an alcohol product of formula (II) as compared to areference polypeptide of SEQ ID NO: 4 or 244, wherein the epoxidesubstrate is cis- or trans-2,3-epoxybutane.

In some embodiments the engineered HHDH polypeptides of the disclosureare capable of an increased rate for converting a non-terminal epoxideto the corresponding alcohol product as compared to a referencepolypeptide of SEQ ID NO: 4 or 244.

In some embodiments the engineered HHDH polypeptides of the disclosureare capable of an increased rate of converting the haloalcohol offormula (II) to the epoxide of formula (I) as compared to a referencepolypeptide of SEQ ID NO: 4 or 244.

In some embodiments the engineered HHDH polypeptides of the disclosureare capable of catalyzing a ring closure reaction converting alcoholsubstrate of formula (II) to an epoxide product of formula (I) with anincreased enantiomeric excess of as compared to a reference polypeptideof SEQ ID NO: 244. In some embodiments of the catalyzed ring closurereaction the alcohol substrate of formula (II) ispara-nitro-styrene-hydroxy-halide (PNSHH) and the epoxide product offormula (I) formed in enantiomeric excess is either R- orS-para-nitro-styrene oxide (PNSO).

In another aspect, the present disclosure provides polynucleotidesencoding any of the engineered HHDH polypeptides, as well as vectors andhost cells comprising the polynucleotides.

In another aspect, the present disclosure provides methods of using theengineered HHDH polypeptides. In some embodiments, the disclosureprovide a method of converting an epoxide of formula (I) to an alcoholof formula (II) (in a reaction scheme as shown below)

wherein, R¹ is a substituted or unsubstituted alkyl, cycloalkyl,heteocycloalkyl, aryl, or heteroaryl; R² is H, or substituted orunsubstituted alkyl, cylcoalkyl, heteocycloalkyl, aryl, or heteroaryl;or wherein R¹ and R² forms a ring; and said method comprises contactingthe epoxide of formula (I) with an engineered HHDH of the disclosure inthe presence of a nucleophile (Nuc⁻) under reaction conditions suitablefor converting the epoxide to the alcohol product.

In certain embodiments, the method can be carried out wherein theepoxide of formula (I) is a terminal epoxide. In some embodiments, themethod can be carried out wherein R¹ is a substituted or unsubstitutedaryl group, or substituted or unsubstituted phenyl group; or, whereinthe epoxide is selected from the group consisting of: styrene oxide;para-nitrostyrene oxide; benzylethylene oxide; 1,2-epoxybutane; and 1,2epoxyhexane; or wherein R¹ and R² forms a ring comprising a substitutedor unsubstituted cycloalkyl or heterocycloalkyl group. In certainembodiments, the method can be carried out wherein the nucleophile(Nuc⁻) is selected from: Br⁻, Cl⁻, I⁻, NO₂ ⁻, N₃ ⁻, CN⁻, OCN⁻, SCN⁻, orformate.

5. DETAILED DESCRIPTION 1.1 Abbreviations

The abbreviations used for the genetically encoded amino acids areconventional and are as follows:

Amino Acid Three-Letter One-Letter Abbreviation Alanine Ala A ArginineArg R Asparagine Asn N Aspartate Asp D Cysteine Cys C Glutamate Glu EGlutamine Gln Q Glycine Gly G Histidine HIS H Isoleucine Ile I LeucineLeu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro PSerine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine ValV

1.2 Definitions

As used herein, the following terms are intended to have the followingmeanings.

“Halohydrin Dehalogenase” and “HHDH” are used interchangeably herein torefer to a polypeptide having an enzymatic capability of converting anepoxide group to a haloalcohol. In some embodiments, the HHDH is alsocapable of converting a haloalcohol to an epoxide. More specifically,the HHDH polypeptides of the invention are capable of stereoselectivelyconverting the compound of formula (I), infra to the correspondingproduct of formula (II), infra. The polypeptide can utilize azide (N₃ ⁻)or another nucleophile, such as Br⁻, Cl⁻, I⁻, NO₂ ⁻, CN⁻, OCN⁻, SCN⁻, orformate (HCOO⁻). Halohydrin dehalogenases as used herein includenaturally occurring (wild type) halohydrin dehalogenases as well asnon-naturally occurring engineered polypeptides generated by humanmanipulation.

“Coding sequence” refers to that portion of a sequence of nucleic acids(e.g., a gene) that encodes an amino acid sequence of a protein.

“Naturally-occurring” or “wild-type” refers to the form found in nature.For example, a naturally occurring or wild-type polypeptide orpolynucleotide sequence is a sequence present in an organism that can beisolated from a source in nature and which has not been intentionallymodified by human manipulation.

“Recombinant” or “engineered” or “non-naturally-occurring” when usedwith reference to, e.g., a cell, nucleic acid sequence, or polypeptide,refers to a material, or a material corresponding to the natural ornative form of the material, that has been modified in a manner thatwould not otherwise exist in nature, or is identical thereto butproduced or derived from synthetic materials and/or by manipulationusing recombinant techniques. Non-limiting examples include, amongothers, recombinant cells expressing genes that are not found within thenative (non-recombinant) form of the cell or express native genes thatare otherwise expressed at a different level.

“Percentage of sequence identity” and “percentage homology” are usedinterchangeably herein to refer to comparisons among polynucleotides andpolypeptides, and are determined by comparing two optimally alignedsequences over a comparison window, wherein the portion of thepolynucleotide or polypeptide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage may be calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison andmultiplying the result by 100 to yield the percentage of sequenceidentity. Alternatively, the percentage may be calculated by determiningthe number of positions at which either the identical nucleic acid baseor amino acid residue occurs in both sequences or a nucleic acid base oramino acid residue is aligned with a gap to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the window of comparison and multiplying the result by100 to yield the percentage of sequence identity. Those of skill in theart appreciate that there are many established algorithms available toalign two sequences. Optimal alignment of sequences for comparison canbe conducted, e.g., by the local homology algorithm of Smith andWaterman, 1981, Adv. Appl. Math. 2:482, by the homology alignmentalgorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, by thesearch for similarity method of Pearson and Lipman, 1988, Proc. Natl.Acad. Sci. USA 85:2444, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG WisconsinSoftware Package), or by visual inspection (see generally, CurrentProtocols in Molecular Biology, F. M. Ausubel et al., eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)). Examples ofalgorithms that are suitable for determining percent sequence identityand sequence similarity are the BLAST and BLAST 2.0 algorithms, whichare described in Altschul et al., 1990, J. Mol. Biol. 215: 403-410 andAltschul et al., 1977, Nucleic Acids Res. 3389-3402, respectively.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information website. This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas, the neighborhood word score threshold (Altschul et al, supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are then extended inboth directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always>0) and N (penalty score formismatching residues; always<0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlength(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff, 1989, Proc Natl Acad Sci USA 89:10915). Exemplarydetermination of sequence alignment and % sequence identity can employthe BESTFIT or GAP programs in the GCG Wisconsin Software package(Accelrys, Madison Wis.), using default parameters provided.

“Reference sequence” refers to a defined sequence used as a basis for asequence comparison. A reference sequence may be a subset of a largersequence, for example, a segment of a full-length gene or polypeptidesequence. Generally, a reference sequence is at least 20 nucleotide oramino acid residues in length, at least 25 residues in length, at least50 residues in length, or the full length of the nucleic acid orpolypeptide. Since two polynucleotides or polypeptides may each (1)comprise a sequence (i.e., a portion of the complete sequence) that issimilar between the two sequences, and (2) may further comprise asequence that is divergent between the two sequences, sequencecomparisons between two (or more) polynucleotides or polypeptide aretypically performed by comparing sequences of the two polynucleotidesover a “comparison window” to identify and compare local regions ofsequence similarity. In some embodiments, a “reference sequence” can bebased on a primary amino acid sequence, where the reference sequence isa sequence that can have one or more changes in the primary sequence.For instance, a “reference sequence based on SEQ ID NO: 4 having at theresidue corresponding to X202 a leucine” refers to a reference sequencein which the residue at a position 202 in SEQ ID NO: 4 has been changedto a leucine. The term “reference sequence” is not intended to belimited to wild-type sequences, and can include engineered sequences.For example, in some embodiments, a “reference sequence” can be thepreviously engineered polypeptide sequences of SEQ ID NO: 4 or SEQ IDNO: 244.

“Comparison window” refers to a conceptual segment of at least about 20contiguous nucleotide positions or amino acids residues wherein asequence may be compared to a reference sequence of at least 20contiguous nucleotides or amino acids and wherein the portion of thesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. The comparison window can be longer than 20contiguous residues, and includes, optionally 30, 40, 50, 100, or longerwindows.

“Substantial identity” refers to a polynucleotide or polypeptidesequence that has at least 80 percent sequence identity, at least 85percent identity and 89 to 95 percent sequence identity, more usually atleast 99 percent sequence identity as compared to a reference sequenceover a comparison window of at least 20 residue positions, frequentlyover a window of at least 30-50 residues, wherein the percentage ofsequence identity is calculated by comparing the reference sequence to asequence that includes deletions or additions which total 20 percent orless of the reference sequence over the window of comparison. Inspecific embodiments applied to polypeptides, the term “substantialidentity” means that two polypeptide sequences, when optimally aligned,such as by the programs GAP or BESTFIT using default gap weights, shareat least 80 percent sequence identity, preferably at least 89 percentsequence identity, at least 95 percent sequence identity or more (e.g.,99 percent sequence identity). Preferably, residue positions which arenot identical differ by conservative amino acid substitutions.

“Corresponding to”, “reference to” or “relative to” when used in thecontext of the numbering of a given amino acid or polynucleotidesequence refers to the numbering of the residues of a specifiedreference sequence when the given amino acid or polynucleotide sequenceis compared to the reference sequence. In other words, the residuenumber or residue position of a given polymer is designated with respectto the reference sequence rather than by the actual numerical positionof the residue within the given amino acid or polynucleotide sequence.For example, a given amino acid sequence, such as that of an engineeredhalohydrin dehalogenase, can be aligned to a reference sequence byintroducing gaps to optimize residue matches between the two sequences.In these cases, although the gaps are present, the numbering of theresidue in the given amino acid or polynucleotide sequence is made withrespect to the reference sequence to which it has been aligned.

“Stereoselectivity” refers to the preferential formation in a chemicalor enzymatic reaction of one stereoisomer over another.Stereoselectivity can be partial, where the formation of onestereoisomer is favored over the other, or it may be complete where onlyone stereoisomer is formed. When the stereoisomers are enantiomers, thestereoselectivity is referred to as enantioselectivity, the fraction(typically reported as a percentage) of one enantiomer in the sum ofboth. It is commonly alternatively reported in the art (typically as apercentage) as the enantiomeric excess (e.e.) calculated therefromaccording to the formula [major enantiomer−minor enantiomer]/[majorenantiomer+minor enantiomer]. Where the stereoisomers arediastereoisomers, the stereoselectivity is referred to asdiastereoselectivity, the fraction (typically reported as a percentage)of one diastereomer in a mixture of two diastereomers, commonlyalternatively reported as the diastereomeric excess (d.e.). Enantiomericexcess and diastereomeric excess are types of stereomeric excess.

“Highly stereoselective” refers to a halohydrin dehalogenase polypeptidethat is capable of converting the substrate to the corresponding producthaving the chemical formula (II) or (III) with at least about 85%stereomeric excess.

“Stereospecificity” refers to the preferential conversion in a chemicalor enzymatic reaction of one stereoisomer over another.Stereospecificity can be partial, where the conversion of onestereoisomer is favored over the other, or it may be complete where onlyone stereoisomer is converted.

“Chemoselectivity” refers to the preferential formation in a chemical orenzymatic reaction of one product over another.

“Regioselectivity” refers to the preference of one direction of bondmaking or breaking over another. For example, nucleophilic attack in theconversion of an epoxide compound of formula (I) infra, to an alcoholcompound of formula (II) infra can be regioselective for either the α orβ-carbon of the epoxide. The α or β-carbon of an epoxide compound areillustrated as follows:

Exemplary epoxide ring-opening products exhibiting α-regioselectivitywould include:

Whereas, exemplary epoxide ring-opening products exhibitingβ-regioselectivity would include:

“Non-terminal epoxide” refers to an epoxide having the followingstructure:

wherein at least one of R^(a) and R^(b) and one of R^(c) and R^(d) is acarbon atom, such as a substituted or unsubstituted alkyl or aryl, andincluding carbon atoms forming a ring, such as in the case of cyclicepoxides.

“Cyclic epoxide” refers to an epoxide of the following structure:

wherein, R^(e) is a carbon ring, for example, substituted orunsubstituted cycloalkyl or heterocycloalkyl. Exemplary carbon rings,include, among others, cyclopentyls, such as cyclopentanyl andcyclopentenyl, and cyclohexanyl and cyclohexenyl. The carbon ring canalso comprise of two rings fused to each other.

“Improved enzyme property” refers to a halohydrin dehalogenasepolypeptide that exhibits an improvement in any enzyme property ascompared to a reference halohydrin dehalogenase. For the engineeredhalohydrin dehalogenase polypeptides described herein, the comparison isgenerally made to the wild-type halohydrin dehalogenase enzyme, althoughin some embodiments, the reference halohydrin dehalogenase can beanother improved engineered halohydrin dehalogenase. Enzyme propertiesfor which improvement is desirable include, but are not limited to,enzymatic activity (which can be expressed in terms of percentconversion of the substrate), thermal stability, solvent stability, pHactivity profile, refractoriness to inhibitors (e.g., productinhibition), stereospecificity, and stereoselectivity (includingenantioselectivity), as well as regioselectivity.

“Increased enzymatic activity” refers to an improved property of theengineered halohydrin dehalogenase polypeptides, which can berepresented by an increase in specific activity (e.g., productproduced/time/weight protein) or an increase in percent conversion ofthe substrate to the product (e.g., percent conversion of startingamount of substrate to product in a specified time period using aspecified amount of HHDH) as compared to the reference halohydrindehalogenase enzyme. Exemplary methods to determine enzyme activity areprovided in the Examples. Any property relating to enzyme activity maybe affected, including the classical enzyme properties of K_(M), V_(max)or k_(cat), changes of which can lead to increased enzymatic activity.Improvements in enzyme activity can be from about 1.5 times theenzymatic activity of the corresponding wild-type halohydrindehalogenase enzyme, to as much as 2 times, 5 times, 10 times, 20 times,25 times, 50 times, 75 times, 100 times, or more enzymatic activity thanthe naturally occurring halohydrin dehalogenase or another engineeredhalohydrin dehalogenase from which the halohydrin dehalogenasepolypeptides were derived. In specific embodiments, the engineeredhalohydrin dehalogenase enzyme exhibits improved enzymatic activity inthe range of 1.5 to 50 times, 1.5 to 100 times greater than that of theparent halohydrin dehalogenase enzyme. It is understood by the skilledartisan that the activity of any enzyme is diffusion limited such thatthe catalytic turnover rate cannot exceed the diffusion rate of thesubstrate. The theoretical maximum of the diffusion limit, ork_(cat)/K_(m), is generally about 10⁸ to 10⁹ (M⁻¹s⁻¹). Hence, anyimprovements in the enzyme activity of the halohydrin dehalogenase willhave an upper limit related to the diffusion rate of the substratesacted on by the halohydrin dehalogenase enzyme. Halohydrin dehalogenaseactivity can be measured by any one of standard assays used formeasuring reaction rates. Comparisons of enzyme activities are madeusing a defined preparation of enzyme, a defined assay under a setcondition, and one or more defined substrates, as further described indetail herein. Generally, when lysates are compared, the numbers ofcells and the amount of protein assayed are determined as well as use ofidentical expression systems and identical host cells to minimizevariations in amount of enzyme produced by the host cells and present inthe lysates.

“Inhibition constant” or “K_(i)” are used interchangeably herein torefer to the inhibition constant of an inhibitor for the enzyme ascommonly understood by those skilled in the art. These include theclassical descriptions of K_(i) for competitive, uncompetitive, andnon-competitive inhibitors.

“Conversion” refers to the enzymatic transformation of the substrate tothe corresponding product. “Percent conversion” refers to the percent ofthe substrate that is converted to the product within a period of timeunder specified conditions. Thus, the “enzymatic activity” or “activity”of a halohydrin dehalogenase polypeptide can be expressed as “percentconversion” of the substrate to the product within a certain timeperiod.

“Thermostable” refers to a halohydrin dehalogenase polypeptide thatmaintains similar activity (more than 60% to 80% for example) afterexposure to elevated temperatures (e.g., 40-80° C.) for a period of time(e.g., 0.5-24 hrs) compared to the untreated enzyme.

“Solvent stable” refers to a halohydrin dehalogenase polypeptide thatmaintains similar activity (more than e.g., 60% to 80%) after exposureto varying concentrations (e.g., 5-99%) of solvent (, tetrahydrofuran,2-methyltetrahydrofuran, toluene, butylacetate, methyl tert-butylether,etc.) for a period of time (e.g., 0.5-24 hrs) compared to the untreatedenzyme.

“pH stable” refers to a halohydrin dehalogenase polypeptide thatmaintains similar activity (more than e.g., 60% to 80%) after exposureto high or low pH (e.g., 4.5-6 or 8 to 12) for a period of time (e.g.,0.5-24 hrs) compared to the untreated enzyme.

“Thermo- and solvent stable” refers to a halohydrin dehalogenasepolypeptide that is both thermostable and solvent stable.

“Derived from” as used herein in the context of engineered halohydrindehalogenase enzymes, identifies the originating halohydrin dehalogenaseenzyme, and/or the gene encoding such halohydrin dehalogenase enzyme,upon which the engineering was based. For example, the engineeredhalohydrin dehalogenase enzyme of SEQ ID NO: 60 was obtained byartificially evolving, over multiple generations the gene encoding theAgrobacterium radiobacter halohydrin dehalogenase enzyme HheC(accession: gi|15213643|gb|AAK92099.1|AF397296_(—)1 halohydrindehalogenase). Thus, this engineered halohydrin dehalogenase enzyme is“derived from” the wild-type HheC halohydrin dehalogenase.

“Hydrophilic amino acid or residue” refers to an amino acid or residuehaving a side chain exhibiting a hydrophobicity of less than zeroaccording to the normalized consensus hydrophobicity scale of Eisenberget al., 1984, J. Mol. Biol. 179:125-142. Genetically encoded hydrophilicamino acids include L-Thr (T), L-Ser (S), L-His (H), L-Glu (E), L-Asn(N), L-Gln (Q), L-Asp (D), L-Lys (K) and L-Arg (R).

“Acidic amino acid or residue” refers to a hydrophilic amino acid orresidue having a side chain exhibiting a pK value of less than about 6when the amino acid is included in a peptide or polypeptide. Acidicamino acids typically have negatively charged side chains atphysiological pH due to loss of a hydrogen ion. Genetically encodedacidic amino acids include L-Glu (E) and L-Asp (D).

“Basic amino acid or residue” refers to a hydrophilic amino acid orresidue having a side chain exhibiting a pK value of greater than about6 when the amino acid is included in a peptide or polypeptide. Basicamino acids typically have positively charged side chains atphysiological pH due to association with hydronium ion. Geneticallyencoded basic amino acids include L-Arg (R) and L-Lys (K).

“Polar amino acid or residue” refers to a hydrophilic amino acid orresidue having a side chain that is uncharged at physiological pH, butwhich has at least one bond in which the pair of electrons shared incommon by two atoms is held more closely by one of the atoms.Genetically encoded polar amino acids include L-Asn (N), L-Gln (Q),L-Ser (S) and L-Thr (T).

“Hydrophobic amino acid or residue” refers to an amino acid or residuehaving a side chain exhibiting a hydrophobicity of greater than zeroaccording to the normalized consensus hydrophobicity scale of Eisenberget al., 1984, J. Mol. Biol. 179:125-142. Genetically encoded hydrophobicamino acids include L-Pro (P), L-Ile (I), L-Phe (F), L-Val (V), L-Leu(L), L-Trp (W), L-Met (M), L-Ala (A) and L-Tyr (Y).

“Aromatic amino acid or residue” refers to a hydrophilic or hydrophobicamino acid or residue having a side chain that includes at least onearomatic or heteroaromatic ring. Genetically encoded aromatic aminoacids include L-Phe (F), L-Tyr (Y) and L-Trp (W). Although owing to thepKa of its heteroaromatic nitrogen atom L-His (H) it is sometimesclassified as a basic residue, or as an aromatic residue as its sidechain includes a heteroaromatic ring, herein histidine is classified asa hydrophilic residue or as a “constrained residue” (see below).

“Constrained amino acid or residue” refers to an amino acid or residuethat has a constrained geometry. Herein, constrained residues includeL-pro (P) and L-his (H). Histidine has a constrained geometry because ithas a relatively small imidazole ring. Proline has a constrainedgeometry because it also has a five membered ring.

“Non-polar amino acid or residue” refers to a hydrophobic amino acid orresidue having a side chain that is uncharged at physiological pH andwhich has bonds in which the pair of electrons shared in common by twoatoms is generally held equally by each of the two atoms (i.e., the sidechain is not polar). Genetically encoded non-polar amino acids includeL-Gly (G), L-Leu (L), L-Val (V), L-Ile (I), L-Met (M) and L-Ala (A).

“Aliphatic amino acid or residue” refers to a hydrophobic amino acid orresidue having an aliphatic hydrocarbon side chain. Genetically encodedaliphatic amino acids include L-Ala (A), L-Val (V), L-Leu (L) and L-Ile(I).

“Cysteine” or L-Cys (C) is unusual in that it can form disulfide bridgeswith other L-Cys (C) amino acids or other sulfanyl- orsulfhydryl-containing amino acids. The “cysteine-like residues” includecysteine and other amino acids that contain sulfhydryl moieties that areavailable for formation of disulfide bridges. The ability of L-Cys (C)(and other amino acids with —SH containing side chains) to exist in apeptide in either the reduced free —SH or oxidized disulfide-bridgedform affects whether L-Cys (C) contributes net hydrophobic orhydrophilic character to a peptide. While L-Cys (C) exhibits ahydrophobicity of 0.29 according to the normalized consensus scale ofEisenberg (Eisenberg et al., 1984, supra), it is to be understood thatfor purposes of the present disclosure L-Cys (C) is categorized into itsown unique group.

“Small amino acid or residue” refers to an amino acid or residue havinga side chain that is composed of a total three or fewer carbon and/orheteroatoms (excluding the α-carbon and hydrogens). The small aminoacids or residues may be further categorized as aliphatic, non-polar,polar or acidic small amino acids or residues, in accordance with theabove definitions. Genetically-encoded small amino acids include L-Ala(A), L-Val (V), L-Cys (C), L-Asn (N), L-Ser (S), L-Thr (T) and L-Asp(D).

“Hydroxyl-containing amino acid or residue” refers to an amino acidcontaining a hydroxyl (—OH) moiety. Genetically-encodedhydroxyl-containing amino acids include L-Ser (S) L-Thr (T) and L-Tyr(Y).

“Amino acid difference” or “residue difference” refers to a change inthe residue at a specified position of a polypeptide sequence whencompared to a reference sequence. For example, a residue difference atposition X139, where the reference sequence has a tryptophan, refers toa change of the residue at position X139 to any residue other thantryptophan. As disclosed herein, an enzyme can include one or moreresidue differences relative to a reference sequence, where multipleresidue differences typically are indicated by a list of the specifiedpositions where changes are made relative to the reference sequence(e.g., “one or more residue differences as compared to SEQ ID NO: 244 atthe following residue positions: X34; X51; X81; X85; X110; X131; X167;X232; X242; X248; and X254.”). Accordingly, a “group of residuedifferences” or “combination of residue differences” refers to a definedset of residue differences (e.g., “one or more groups of residuedifferences selected from: X139 is M, and X176 is S; X139 is M, and X176is T; and X139 is S, and X176 is T.”).

“Conservative” amino acid substitutions or mutations refer to theinterchangeability of residues having similar side chains, and thustypically involves substitution of the amino acid in the polypeptidewith amino acids within the same or similar defined class of aminoacids. However, as used herein, in some embodiments, conservativemutations do not include substitutions from a hydrophilic tohydrophilic, hydrophobic to hydrophobic, hydroxyl-containing tohydroxyl-containing, or small to small residue, if the conservativemutation can instead be a substitution from an aliphatic to analiphatic, non-polar to non-polar, polar to polar, acidic to acidic,basic to basic, aromatic to aromatic, or constrained to constrainedresidue. Further, as used herein, A, V, L, or I can be conservativelymutated to either another aliphatic residue or to another non-polarresidue. Table 1 below shows exemplary conservative substitutions.

TABLE 1 Residue Possible Conservative Mutations A, L, V, I Otheraliphatic (A, L, V, I) Other non-polar (A, L, V, I, G, M) G, M Othernon-polar (A, L, V, I, G, M) D, E Other acidic (D, E) K, R Other basic(K, R) P, H Other constrained (P, H) N, Q, S, T Other polar Y, W, FOther aromatic (Y, W, F) C None

“Non-conservative substitution” refers to substitution or mutation of anamino acid in the polypeptide with an amino acid with significantlydiffering side chain properties. Non-conservative substitutions may useamino acids between, rather than within, the defined groups listedabove. In one embodiment, a non-conservative mutation affects (a) thestructure of the peptide backbone in the area of the substitution (e.g.,proline for glycine) (b) the charge or hydrophobicity, or (c) the bulkof the side chain.

“Deletion” refers to modification to the polypeptide by removal of oneor more amino acids from the reference polypeptide. Deletions cancomprise removal of 1 or more amino acids, 2 or more amino acids, 5 ormore amino acids, 10 or more amino acids, 15 or more amino acids, or 20or more amino acids, up to 10% of the total number of amino acids, up to20% of the total number of amino acids, or up to 30% of the total numberof amino acids making up the polypeptide while retaining enzymaticactivity and/or retaining the improved properties of an engineeredhalohydrin dehalogenase enzyme. Deletions can be directed to theinternal portions and/or terminal portions of the polypeptide. Invarious embodiments, the deletion can comprise a continuous segment orcan be discontinuous.

“Insertion” refers to modification to the polypeptide by addition of oneor more amino acids from the reference polypeptide. In some embodiments,the improved engineered halohydrin dehalogenase enzymes compriseinsertions of one or more amino acids to the naturally occurringhalohydrin dehalogenase polypeptide as well as insertions of one or moreamino acids to other improved halohydrin dehalogenase polypeptides.Insertions can be in the internal portions of the polypeptide, or to thecarboxy or amino terminus. Insertions as used herein include fusionproteins as is known in the art. The insertion can be a contiguoussegment of amino acids or separated by one or more of the amino acids inthe naturally occurring polypeptide.

“Fragment” as used herein refers to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion, but where the remainingamino acid sequence is identical to the corresponding positions in thesequence. Fragments can be at least 14 amino acids long, at least 20amino acids long, at least 50 amino acids long or longer, and up to 70%,80%, 90%, 95%, 98%, and 99% of the full-length halohydrin dehalogenasepolypeptide, for example the polypeptide of SEQ ID NO:2, 4, or 86.

“Isolated polypeptide” refers to a polypeptide which is substantiallyseparated from other contaminants that naturally accompany it, e.g.,protein, lipids, and polynucleotides. The term embraces polypeptideswhich have been removed or purified from their naturally-occurringenvironment or expression system (e.g., host cell or in vitrosynthesis). The improved halohydrin dehalogenase enzymes may be presentwithin a cell, present in the cellular medium, or prepared in variousforms, such as lysates or isolated preparations. As such, in someembodiments, the improved halohydrin dehalogenase enzyme can be anisolated polypeptide.

“Substantially pure polypeptide” refers to a composition in which thepolypeptide species is the predominant species present (i.e., on a molaror weight basis it is more abundant than any other individualmacromolecular species in the composition), and is generally asubstantially purified composition when the object species comprises atleast about 50 percent of the macromolecular species present by mole or% weight. Generally, a substantially pure halohydrin dehalogenasecomposition will comprise about 60% or more, about 70% or more, about80% or more, about 90% or more, about 95% or more, and about 98% or moreof all macromolecular species by mole or % weight present in thecomposition. In some embodiments, the object species is purified toessential homogeneity (i.e., contaminant species cannot be detected inthe composition by conventional detection methods) wherein thecomposition consists essentially of a single macromolecular species.Solvent species, small molecules (<500 Daltons), and elemental ionspecies are not considered macromolecular species. In some embodiments,the isolated improved halohydrin dehalogenases polypeptide is asubstantially pure polypeptide composition.

“Stringent hybridization” is used herein to refer to conditions underwhich nucleic acid hybrids are stable. As known to those of skill in theart, the stability of hybrids is reflected in the melting temperature(T_(m)) of the hybrids. In general, the stability of a hybrid is afunction of ion strength, temperature, G/C content, and the presence ofchaotropic agents. The T_(m) values for polynucleotides can becalculated using known methods for predicting melting temperatures (see,e.g., Baldino et al., Methods Enzymology 168:761-777; Bolton et al.,1962, Proc. Natl. Acad. Sci. USA 48:1390; Bresslauer et al., 1986, Proc.Natl. Acad. Sci. USA 83:8893-8897; Freier et al., 1986, Proc. Natl.Acad. Sci. USA 83:9373-9377; Kierzek et al., Biochemistry 25:7840-7846;Rychlik et al., 1990, Nucleic Acids Res 18:6409-6412 (erratum, 1991,Nucleic Acids Res 19:698); Sambrook et al., supra); Suggs et al., 1981,In Developmental Biology Using Purified Genes (Brown et al., eds.), pp.683-693, Academic Press; and Wetmur, 1991, Crit. Rev Biochem Mol Biol26:227-259. All publications incorporate herein by reference). In someembodiments, the polynucleotide encodes the polypeptide disclosed hereinand hybridizes under defined conditions, such as moderately stringent orhighly stringent conditions, to the complement of a sequence encoding anengineered halohydrin dehalogenase enzyme of the present disclosure.

“Hybridization stringency” relates to hybridization conditions, such aswashing conditions, in the hybridization of nucleic acids. Generally,hybridization reactions are performed under conditions of lowerstringency, followed by washes of varying but higher stringency. Theterm “moderately stringent hybridization” refers to conditions thatpermit target-DNA to bind a complementary nucleic acid that has about60% identity, preferably about 75% identity, about 85% identity to thetarget DNA; with greater than about 90% identity totarget-polynucleotide. Exemplary moderately stringent conditions areconditions equivalent to hybridization in 50% formamide, 5× Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE,0.2% SDS, at 42° C. “High stringency hybridization” refers generally toconditions that are about 10° C. or less from the thermal meltingtemperature T_(m) as determined under the solution condition for adefined polynucleotide sequence. In some embodiments, a high stringencycondition refers to conditions that permit hybridization of only thosenucleic acid sequences that form stable hybrids in 0.018M NaCl at 65° C.(i.e., if a hybrid is not stable in 0.018M NaCl at 65° C., it will notbe stable under high stringency conditions, as contemplated herein).High stringency conditions can be provided, for example, byhybridization in conditions equivalent to 50% formamide, 5×Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE,and 0.1% SDS at 65° C. Another high stringency condition is hybridizingin conditions equivalent to hybridizing in 5×SSC containing 0.1% (w:v)SDS at 65° C. and washing in 0.1×SSC containing 0.1% SDS at 65° C. Otherhigh stringency hybridization conditions, as well as moderatelystringent conditions, are described in the references cited above.

“Heterologous” polynucleotide refers to any polynucleotide that isintroduced into a host cell by laboratory techniques, and includespolynucleotides that are removed from a host cell, subjected tolaboratory manipulation, and then reintroduced into a host cell.

“Codon optimized” refers to changes in the codons of the polynucleotideencoding a protein to those preferentially used in a particular organismsuch that the encoded protein is efficiently expressed in the organismof interest. Although the genetic code is degenerate in that most aminoacids are represented by several codons, called “synonyms” or“synonymous” codons, it is well known that codon usage by particularorganisms is nonrandom and biased towards particular codon triplets.This codon usage bias may be higher in reference to a given gene, genesof common function or ancestral origin, highly expressed proteins versuslow copy number proteins, and the aggregate protein coding regions of anorganism's genome. In some embodiments, the polynucleotides encoding thehalohydrin dehalogenases enzymes may be codon optimized for optimalproduction from the host organism selected for expression.

“Preferred, optimal, high codon usage bias codons” refersinterchangeably to codons that are used at higher frequency in theprotein coding regions than other codons that code for the same aminoacid. The preferred codons may be determined in relation to codon usagein a single gene, a set of genes of common function or origin, highlyexpressed genes, the codon frequency in the aggregate protein codingregions of the whole organism, codon frequency in the aggregate proteincoding regions of related organisms, or combinations thereof. Codonswhose frequency increases with the level of gene expression aretypically optimal codons for expression. A variety of methods are knownfor determining the codon frequency (e.g., codon usage, relativesynonymous codon usage) and codon preference in specific organisms,including multivariate analysis, for example, using cluster analysis orcorrespondence analysis, and the effective number of codons used in agene (see GCG CodonPreference, Genetics Computer Group WisconsinPackage; CodonW, John Peden, University of Nottingham; McInerney, J. O,1998, Bioinformatics 14:372-73; Stenico et al., 1994, Nucleic Acids Res.222437-46; Wright, F., 1990, Gene 87:23-29). Codon usage tables areavailable for a growing list of organisms (see for example, Wada et al.,1992, Nucleic Acids Res. 20:2111-2118; Nakamura et al., 2000, Nucl.Acids Res. 28:292; Duret, et al., supra; Henaut and Danchin,“Escherichia coli and Salmonella,” 1996, Neidhardt, et al. Eds., ASMPress, Washington D.C., p. 2047-2066. The data source for obtainingcodon usage may rely on any available nucleotide sequence capable ofcoding for a protein. These data sets include nucleic acid sequencesactually known to encode expressed proteins (e.g., complete proteincoding sequences-CDS), expressed sequence tags (ESTS), or predictedcoding regions of genomic sequences (see for example, Mount, D.,Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Uberbacher, E.C., 1996, Methods Enzymol. 266:259-281; Tiwari et al., 1997, Comput.Appl. Biosci. 13:263-270).

“Control sequence” is defined herein to include all components, whichare necessary or advantageous for the expression of a polypeptide of thepresent disclosure. Each control sequence may be native or foreign tothe nucleic acid sequence encoding the polypeptide. Such controlsequences include, but are not limited to, a leader, polyadenylationsequence, propeptide sequence, promoter, signal peptide sequence, andtranscription terminator.

“Operably linked” is defined herein as a configuration in which acontrol sequence is appropriately placed (i.e., in a functionalrelationship) at a position relative to a polynucleotide of interestsuch that the control sequence directs or regulates the expression ofthe polynucleotide and/or polypeptide of interest.

“Promoter sequence” is a nucleic acid sequence that is recognized by ahost cell for expression of a polynucleotide of interest, such as acoding sequence. The control sequence may comprise an appropriatepromoter sequence. The promoter sequence contains transcriptionalcontrol sequences, which mediate the expression of a polynucleotide ofinterest. The promoter may be any nucleic acid sequence which showstranscriptional activity in the host cell of choice including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

1.3 Engineered Halohydrin Dehalogenase Polypeptides

As discussed above, halohydrin dehalogenases are a class of enzymescapable of mediating the following reaction (Scheme 4):

wherein, the nucleophile (Nuc⁻) can be, among others, Br⁻, Cl⁻, I⁻, NO₂⁻, N₃ ⁻, CN⁻, OCN⁻, SCN⁻, or formate (HCOO⁻). For the wild type A.radiobacter HheC, the enzyme acts generally on epoxides where R² is Hand R¹ is an alkyl or aryl group. The wild type HheC showsR-stereoselectivity for the alcohol product, regioselectivity for ringopening of styrene oxides mainly at the β position, and a preference forring opening of terminal epoxides and ring closure of haloalcoholstowards terminal epoxides. A. radiobacter HheC is also stronglyinhibited by halide, which is a disadvantage where an efficient ringclosure of haloalcohol to epoxide is needed or in processes wherehalides are present for other reasons.

Accordingly, provided in the present disclosure are engineeredhalohydrin dehalogenase (HHDH) polypeptides having altered or improvedenzyme properties, including, among others, changes to substraterecognition, enantioselectivity, halide ion resistance, andregioselectivity. These changes allow the engineered HHDHs to be used inbiocatalytic applications for preparing compounds not efficientlyproduced by other HHDH polypeptides.

In one aspect, engineered HHDH polypeptides comprise an amino acidsequence that differs at one or more residue positions from that of wildtype A. radiobacter HheC or an engineered HHDH polypeptide of SEQ ID NO:4 or SEQ ID NO: 244 to result in the altered or improved enzymeproperty, as further described below. In some embodiments, therecombinant HHDH polypeptides can comprise an amino acid sequence thatis at least 60%, 65%, 70%, 71%, 72%, 73%, 74% 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85% 86% 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence ofSEQ ID NO:4 or SEQ ID NO:244.

In some embodiments, the residue differences can be at one or moreresidue positions selected from X34; X51; X81; X85; X110; X131; X167;X232; X242; X248, and X254. In some embodiments, the residue differencesfor the engineered HHDH polypeptides can be selected from one or more ofthe following: X34 is G; X51 is P; X81 is E, F, M, W, or Y; X85 is V;X110 is S; X131 is A; X167 is H; X232 is S, X242 is A; X248 is V; andX254 is N. As will be understood by those skilled in the art, theindicated amino acids can be replaced by amino acids that areconservative substitutions to effect the changes to the desired enzymeproperty. In some embodiments, the recombinant HHDH polypeptides canhave one or more residue differences at other residue positions. In someembodiment, the differences can be 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-13, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24,1-26, 1-28, 1-30, 1-32, 1-34, 1-36, 1-38, 1-40, 1-42, 1-44, 1-46, 1-48,or 1-50 residue differences at other residue positions as compared toSEQ ID NO: 4 or SEQ ID NO:244. In some embodiment, the differences canbe 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 22,24, 26, 28 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 residuedifferences at other residue positions as compared to SEQ ID NO: 4 orSEQ ID NO:244. In some embodiments, the residue differences at otherresidue positions can comprise conservative mutations.

In some embodiments, the engineered HHDH polypeptides can comprise anamino acid sequence that is at least 60%, 65%, 70%, 71%, 72%, 73%, 74%75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85% 86% 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical tothe amino acid sequence of SEQ ID NO:4 or SEQ ID NO:244 and have one ormore of the following residues: X12 is M, or S; X34 is G; X51 is P; X81is E, F, M, W, or Y; X82 is C, G, K, L, M, N, or W; X83 is C or E; X84is D; X85 is V; X86 is A or H; X110 is S, X131 is A; X134 is C, F, I, K,L, M, or V; X139 is G, I, L, M, P, S, T, or V; X142 is C, M or N; X146is P; X167 is H; X175 is C, G, I, L, M, N, or V; X176 is F, M, Q, or T;X177 is L, S or T; X178 is T; X182 is S; X186 is H; X187 is G, I, or S;X201 is R; X232 is S, X242 is A; X243 is C or L; X245 is S; X246 is S;X247 is N; X248 is V; X249 is A, G, H, I, or S; X252 is C; and X254 isN. In some embodiments, the specified amino acid residues can bereplaced with amino acids that are conservative substitutions of thespecified amino acids. In some embodiments, the recombinant HHDHpolypeptides can have one or more residue differences at other residuepositions. In some embodiment, the differences can be 1-2, 1-3, 1-4,1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14, 1-15, 1-16, 1-18,1-20, 1-22, 1-24, 1-26, 1-28, 1-30, 1-32, 1-34, 1-36, 1-38, 1-40, 1-42,1-44, 1-46, 1-48, or 1-50 residue differences at other residue positionsas compared to SEQ ID NO: 4 or SEQ ID NO:244. In some embodiment, thedifferences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or50 residue differences at other residue positions as compared to SEQ IDNO: 4 or SEQ ID NO:244. In some embodiments, the residue differences atother residue positions can comprise conservative mutations.

In some embodiments, the engineered HHDH polypeptides of the disclosuredescribed above can be combined with residue differences at otherresidue positions to effect changes in the properties of the engineeredHHDH polypeptide. In some embodiments, the residues can be based on theengineered enzymes described in US application publication US2005/0153417, US application publication US2005/0272064, and USapplication publication US 2006/0099700, each of which is herebyincorporated herein by reference. In some embodiments, the amino acidsthat can be used in combination with the residues described aboveinclude, among others, one or more of the following: X2 is G; X4 is S,or V; X8 is K; X10 is L, N, or R; X12 is G, or Y; X20 is C; X21 is M;X30 is V; X38 is K, or L; X39 is G; X41 is A; X42 is A; X52 is K, or T;X54 is I, or V; X58 is G; X60 is T, or V; X63 is V; X67 is N; X68 is G;X69 is R, or V; X70 is F; X72 is Q, or R; X75 is I; X82 is F; X82 is I,S, T, V, or Y; X83 is A; X84 is L; X86 is F; X87 is Q; X91 is E, or R;X95 is A, or G; X99 is G, or N; X100 is A, or M; X107 is R; X112 is A;X113 is S; X116 is V; X117 is P, or T; X121 is D, E, or R; X124 is G;X128 is V; X129 is L; X134 is T; X135 is L, or S; X137 is W; X139 is D,H, K, N, R, or Y; X142 is G, or I; X146 is A, T, or V; X150 is V; X152is T; X174 is G; X175 is S; X176 is D, E, H, R, S; X177 is A, F, G, N;X178 is C, M, V; X179 is D; X180 is T; X181 is E; X182 is N; X184 is K;X186 is W; X187 is H; X189 is T; X194 is I; X195 is N; X196 is L; X200is T; X201 is W; X202 is L; X203 is K; X204 is R; X205 is Y; X215 is E,R; X222 is A; X223 is L; X230 is N; X238 is C, L, R, or T; X240 is T;X241 is A; X243 is S; X245 is A; X246 is V; X247 is D; X249 is R, Y,or * (sequence truncated after X248); X251 is A, or E; and X252 is I, N,or V.

In some embodiments, the HHDH polypeptides can have increasedenantioselectivity for the S-alcohol product as compared to a referenceHHDH polypeptide of SEQ ID NO: 244. In contrast, the reference HHDHpolypeptide of SEQ ID NO: 244 displays R-enantioselectivity, similar tothe wild-type HHDH HheC and the wild-type double-mutant engineered HHDHof SEQ ID NO: 4. In some embodiments, the engineered HHDH polypeptide iscapable of converting a racemic styrene oxide to the correspondingS-alcohol in enantiomeric excess over the R-alcohol as shown below inScheme 5.

In some embodiments, the HHDH polypeptide is capable of convertingracemic styrene oxide to S-azido alcohol in enantiomeric excess in thepresence of the nucleophile N₃. In some embodiments, the HHDHpolypeptide can convert racemic para-nitro-styrene-oxide (PNSO) to thecorresponding S-alcohol in presence of a nucleophile.

In some embodiments, the engineered HHDH polypeptide havingS-enantioselectivity for the reaction of Scheme 5 comprises an aminoacid sequence having one or more of the following residues or groups ofresidues: X243 is L; X99 is N and X139 is M; X85 is V and X243 is S;X146 is P, X176 is S, and X243 is S; X139 is M and X243 is S; X139 is T,X176 is D, and X243 is S; X139 is T, X180 is T, and X243 is S; X139 is Sand X243 is S; X80 is V and X139 is S; X95 is G and X139 is M and X243is S; X121 is R and X176 is T and X243 is S; X62 is V and X139 is M andX197 is G; X112 is A and X139 is S; X139 is S or M; X139 is T and X201is R; X85 is D and X139 is M and X 168 is V. Exemplary polypeptides withS-enantioselectivity include, but are not limited to, polypeptidescomprising amino acid sequences corresponding to SEQ ID NO: 40, 120,122, 130, 132, 136, 140, 144, 158, 162, 166, 274, 276, 286, 288, 290,292, or 294.

In some embodiments, the engineered HHDH are capable of producing theS-alcohol in the reaction of Scheme 5 in at least about 20%, 40%, 60%,or 80% e.e. or more.

In some embodiments, the engineered HHDH capable of producing at leastabout 20% e.e. of the S-alcohol in the reaction of Scheme 5 comprises anamino acid sequence having one or more of the following residues orgroups of residues: X80 is V and X139 is S; X95 is G, X139 is M and X243is S; X243 is L; X121 is R, X176 is T and X243 is S; X139 is M and X243is S; X62 is V, X139 is M and X197 is G; X112 is A and X139 is S; X139is S; X139 is T and X201 is R; X139 is T, X176 is D, and X243 is S; X139is T, X180 is T, and X243 is S; X146 is P, X176 is S, and X243 is S;X139 is S, and X243 is S; X99 is N and X139 is M; X85 is D, X139 is Mand X168 is V; X85 is V and X243 is S; or X139 is M. Exemplarypolypeptides capable of producing at least about 20% e.e. of theS-alcohol include, but are not limited to, polypeptides comprising aminoacid sequences corresponding to SEQ ID NO: 120, 122, 140, 274, 288, 40,162, 166, 276, 292, 294, 136, 158, 130, 132, 286, or 290.

In some embodiments, the engineered HHDH capable of producing at leastabout 40% e.e. of the S-alcohol in the reaction of Scheme 5 comprises anamino acid sequence having one or more of the following residues orgroups of residues: X62 is V, X139 is M and X197 is G; X112 is A andX139 is S; X139 is S; X139 is T and X201 is R; X139 is T, X176 is D, andX243 is S; X139 is T, X180 is T, and X243 is S; X146 is P, X176 is S,and X243 is S; X139 is S, and X243 is S; X99 is N and X139 is M; X85 isD, X139 is M and X168 is V; X85 is V and X243 is S; or X139 is M.Exemplary polypeptides capable of producing at least about 40% e.e. ofthe S-alcohol include, but are not limited to, polypeptides comprisingamino acid sequences corresponding to SEQ ID NO: 40, 162, 166, 276, 292,294, 136, 158, 130, 132, 286, or 290.

In some embodiments, the engineered HHDH capable of producing at leastabout 60% e.e. of the S-alcohol in the reaction of Scheme 5 comprises anamino acid sequence having one or more of the following residues orgroups of residues: X146 is P, X176 is S, and X243 is S; X139 is S, andX243 is S; X99 is N and X139 is M; X85 is D, X139 is M and X168 is V;X85 is V and X243 is S; or X139 is M. Exemplary polypeptides capable ofproducing at least about 60% e.e. of the S-alcohol include, but are notlimited to, a polypeptide comprising an amino acid sequencecorresponding to SEQ ID NO: 136, 158, 130, 132, 286, or 290.

In some embodiments, the engineered HHDH capable of producing at leastabout 80% e.e. of the S-alcohol in the reaction of Scheme 5 comprises anamino acid sequence having one or more of the following residues orgroups of residues: X99 is N and X139 is M; X85 is D, X139 is M and X168is V; X85 is V and X243 is S; or X139 is M. Exemplary polypeptidescapable of producing at least about 80% e.e. of the S-alcohol include,but are not limited to, a polypeptide comprising amino acid sequencecorresponding to SEQ ID NO: 130, 132, 286, or 290.

In some embodiments, the engineered HHDH polypeptide is capable ofimproved production of the R-alcohol in the reaction of Scheme 5 ascompared to the HHDH polypeptide of SEQ ID NO: 244. In some embodiments,the engineered HHDH polypeptide is capable of producing at least about40%, 60%, 80% or even higher % e.e. of the R-alcohol. In someembodiments, the engineered HHDH polypeptide with improvedR-enantioselectivity comprises an amino acid sequence having one or moreof the following residue differences or groups of residue differences ascompared to SEQ ID NO: 244: N176T; N139S/N176T; E33G/W139T/N176S;G25C/W139M/N176T; N113S/W139T/N176S; N176S; W139M/1765/H201R/K204R;W139M/N176S; W139M/N176S/F243L; W139M/N176T; W139S/N176S; W139T/N176S;or T3S/W139M/N176S. Exemplary engineered HHDH polypeptides with thisproperty include, but are not limited to, a polypeptide comprising anamino acid sequence corresponding to SEQ ID NO: 116, 142, 146, 148, 154,156, 268, 270, 272, 278, 282, or 284.

In some embodiments, the engineered HHDH polypeptide is capable ofconverting racemic styrene oxide to the corresponding R-alcohol ingreater enantiomeric excess over the S-alcohol than HHDH polypeptide ofSEQ ID NO: 244. In some embodiments, the HHDH polypeptide can convertracemic para-nitro-styrene-oxide to R-para-nitro-hydroxy-halide ingreater enantiomeric excess to the S-para-nitro-hydroxy-halide ingreater enantiomeric excess than a halohydrin dehalogenase polypeptideof SEQ ID NO: 244.

In some embodiments, the engineered HHDH polypeptide is capable ofproducing at least about 40% e.e. of the R-alcohol comprises an aminoacid sequence having one or more of the following residue differences orgroups of residue differences: E33G/W139T/N176S; G25C/W139M/N176T;N113S/W139T/N176S; W139M/1765/H201R/K204R; W139M/N176S;W139M/N176S/F243L; W139M/N176T; W139S/N176S; W139T/N176S; or N176T;W139S/N176T. Exemplary polypeptides capable of producing at least about40% e.e. of the R-alcohol include, but are not limited to, a polypeptidecomprising amino acid sequence corresponding to SEQ ID NO: 116, 142,146, 148, 154, 156, 270, 272, 278, 282, or 284.

In some embodiments, the engineered HHDH polypeptide capable ofproducing at least about 60% e.e. of the R-alcohol comprises an aminoacid sequence having one or more of the following residues or groups ofresidues: E33G/W139T/N176S; G25C/W139M/N176T; N113S/W139T/N176S;W139M/1765/H201R/K204R; W139M/N176S; W139M/N176S/F243L; W139M/N176T;W139S/N176S; or W139T/N176S. Exemplary polypeptides capable of producingat least about 60% e.e. of the R-alcohol include, but are not limitedto, a polypeptide comprising amino acid sequence corresponding to SEQ IDNO: 116, 146, 148, 154, 156, 272, 278, 282, or 284.

In some embodiments, the engineered HHDH polypeptide capable ofproducing at least about 80% e.e. of the R-alcohol comprises an aminoacid sequence having a serine at position 3, a methionine at position139, and a serine at position 176. By way of example, a polypeptidecapable of producing at least about 80% e.e. of the R-alcohol comprisesthe amino acid sequence of SEQ ID NO: 282.

In some embodiments, the engineered HHDH is capable of converting aracemic mixture of para nitro styrene haloalcohol (wherein X is F⁻, Cl⁻,Br⁻, or I⁻) to S-para-nitro-styrene-oxide as shown in Scheme 6.

In some embodiments the engineered HHDH is capable of converting aracemic para nitro styrene haloalcohol to S-para-nitro-styrene-oxidecomprises one or more residues or groups of residues selected from: X139is T; X139 is T and X249 is H; X139 is S and X249 is V; X139 is M andX223 is V, and X249 is P. Exemplary polypeptides with this propertyinclude, but are not limited to, polypeptides comprising an amino acidsequence corresponding to SEQ ID NO:260, 262, 264, or 266.

In some embodiments, the engineered HHDH is capable of converting aracemic mixture of para nitro styrene haloalcohol toR-para-nitro-styrene-oxide, with improved R-enantioselectivity comparedto the polypeptide of SEQ ID NO: 244. In some embodiments, theengineered HHDH capable of at least about 10-fold improvedR-enantioselectivity and comprises one, or more residues or groups ofresidues selected from: X114 is V and X178 is C; X175 is G, L, M, or V;X175 is L and X222 is A. Exemplary polypeptides with this propertyinclude, but are not limited to, polypeptides comprising an amino acidsequence corresponding to SEQ ID NO: 44, 46, 48, 50, 86, 248, or 256. Insome embodiments, the engineered HHDH capable of at least about 100-foldimproved R-enantioselectivity and comprises one, or more residues orgroups of residues selected from: X175 is G, L, M, or V; X175 is L andX222 is A. Exemplary polypeptides with this property include, but arenot limited to, polypeptides comprising an amino acid sequencecorresponding to SEQ ID NO: 44, 46, 48, 50, 86, or 248.

In some embodiments, the engineered HHDH polypeptide can have alteredregioselectivity for the conversion of an epoxide substrate of formula(I) to an alcohol of formula (II) as compared to the wild-type HHDH HheC(accession: gi|15213643|gb|AAK92099.1|AF397296_(—)1 halohydrindehalogenase) or an engineered HHDH polypeptide of SEQ ID NO: 4 or 244.As discussed above, wild-type HheC and the engineered polypeptide of SEQID NO: 4 are regioselective for the β-carbon on the substituted epoxidering. The polypeptide of SEQ ID NO: 244 is about 1 to 2-fold moreregioselective for the α-carbon. In the embodiments herein, theengineered HHDH polypeptides can have increased regioselectivity for theβ-carbon or increased regioselectivity for the α-carbon as compared tothe reference HHDH polypeptides of SEQ ID NO: 4 or SEQ ID NO: 244.

In some embodiments, the engineered HHDH polypeptides of the disclosurehave greater α-regioselectivity than a halohydrin dehalogenasepolypeptide of SEQ ID NO: 244 or SEQ ID NO: 4. In some embodiments, theHHDH polypeptide is capable of at least about 2, 3, 4, 5, 6, or 7-foldmore α than β regioselectivity (having a ratio of α:β>1) in the ringopening of epoxide substrates, wherein the ratio of α to β representsthe increased frequency of nucleophilic attack at the α position versusthe β position.

In some embodiments, the engineered HHDH is capable of convertingstyrene oxide to the S-alcohol in a α-attack and is enantioselective forthe S-enantiomer in the reaction of Scheme 7 below.

Residue differences (as compared to SEQ ID NO: 244) associated withincreased α-attack and enantioselectivity for the S-enantiomer in thereaction of Scheme 7 include: W139L or W139M.

In some embodiments, the engineered HHDH is capable of convertingstyrene oxide to the R-alcohol in an α-attack and is enantioselectivefor the R-enantiomer in the reaction of Scheme 8 below.

Residue differences (as compared to SEQ ID NO: 244) associated withincreased α-attack and enantioselectivity for the R-enantiomer includethe combination of: W139L and P175L. A skilled artisan can determine theresidue differences or residue difference combinations resulting in S orR enantiomer from an α-attack using the guidance provided in the presentdisclosure.

In some embodiments, the engineered HHDH polypeptide is capable of atleast about 3-fold more α than β regioselectivity as compared to theengineered HHDH polypeptide of SEQ ID NO: 244 and comprises an aminoacid sequence having one or more of the residue differences or groups ofresidue differences (as compared to SEQ ID NO: 244) selected from:W139M; D99N/W139M; E85D/W139M/1168V; L62V/W139M/E197G; W139S;V112A/W139S; or D80V/W139S. Exemplary polypeptides with this propertyinclude, but are not limited to, polypeptides comprising amino acidsequences corresponding to SEQ ID NO: 40, 132, 274, 276, 286, 290, or294.

In one embodiment, the engineered HHDH polypeptide is capable of atleast about 6-fold more α than β regioselectivity as compared to theengineered HHDH polypeptide of SEQ ID NO:244 and comprises an amino acidsequence of SEQ ID NO: 132 wherein the residue at position X99 is N andthe residue at position X139 is M.

In one embodiment, the engineered HHDH polypeptide is capable of atleast about 7-fold more α than β regioselectivity as compared to theengineered HHDH polypeptide of SEQ ID NO:244 and comprises an amino acidsequence of SEQ ID NO:286 wherein the residue at position X139 is M.

In some embodiments, the engineered HHDH polypeptide can mediate styreneoxide ring opening by nucleophilic α-attack of azide. In theseembodiments, the HHDH polypeptide can comprise an amino acid sequencehaving one or more of the following residues: W139L, W139M, P175L,N176D, W139S, W139M/F243S, or F243L. Exemplary polypeptides with thisproperty include, but are not limited to, polypeptides comprising aminoacid sequences corresponding to SEQ ID NO: 34, 40, or 122.

In one embodiment, the engineered HHDH polypeptide comprises an aminoacid sequence of SEQ ID NO: 4 or 244 in which the residue at position139 is substituted with a methionine, and having more than 7-fold moreα-attack than β-attack.

In some embodiments, the engineered HHDH polypeptide has increasedβ-selectivity for the conversion of the epoxide of formula (I) to thealcohol of formula (II) than the HHDH polypeptide of SEQ ID NO:244.

In some embodiments, the engineered HHDH is capable of convertingstyrene oxide to the S alcohol in a β attack and enantioselective forthe R-enantiomer in the reaction of Scheme 9 below.

Residue differences (as compared to SEQ ID NO: 244) associated withincreased β attack and enantioselectivity for the R-enantiomer includeN176S.

In some embodiments, the engineered HHDH is capable of convertingstyrene oxide to the S alcohol in a β attack and enantioselective forthe S-enantiomer in the reaction of Scheme 10 below.

Residue differences (as compared to SEQ ID NO: 244) associated withincreased β attack and enantioselectivity for the R-enantiomer includeF243S. A skilled artisan can determine the residue or residuecombinations resulting in S or R enantiomer from a beta attack using theguidance in the present disclosure.

In some embodiments, the engineered HHDH polypeptide is capable of atleast about 2-fold more β than α regioselectivity as compared to theengineered HHDH polypeptide of SEQ ID NO:244, and comprises an aminoacid sequence having one or more residue differences or groups ofresidue differences selected from: X3 is S, X139 is M, and X176 is S;X25 is C, X139 is M, and X176 is T; X113 is S, X139 is T, X176 is S;X139 is S, X176 is S; X139 is M, X176 is S, and X243 is L; X139 is M,X176 is S, X201 is R, and X204 is R; X139 is T, X176 is D, and X243 isS; X139 is M, and X176 is D; X139 is T, X180 is T, and X243 is S; X139is M, and X176 is S; X139 is S, and X176 is T; X139 is T, and X176 is D;and X139 is T, and X176 is S. Exemplary engineered HHDH polypeptideswith increased b selectivity can comprise an amino acid sequencecorresponding to SEQ ID NO: 116, 146, 154, 156, 162, 164, 166, 268, 270,272, 280, 282, or 284.

In some embodiments, the engineered HHDH polypeptide is capable of atleast about 3-fold more β than α regioselectivity than a halohydrindehalogenase polypeptide of SEQ ID NO:244, and comprises an amino acidsequence having one or more residues selected from the following:W139M/N176S/F243L; or W139M/N176D. Exemplary engineered HHDHpolypeptides with increased b selectivity can comprise an amino acidsequence corresponding to SEQ ID NO: 154 or 164.

As discussed above, the wild-type HHDH HheC and engineered HheCdouble-mutant of SEQ ID NO: 4 is limited to ring opening of terminalepoxide substrates or ring closure of haloalcohols to the terminalepoxides. For example, Majeric-Elenkov et al., 2006, Adv. Synth. Catal.348, 579-585 shows that HheA, HheB, and HheC did not show detectableactivity on cyclohexene oxide and 2,3 epoxybutane. In one aspect, thepresent invention provides engineered HHDH polypeptides havingmeasurable and increased rate for converting a non-terminal epoxide tothe corresponding alcohol product as compared to a reference HHDHpolypeptide having the amino acid sequence of SEQ ID NO:4 and SEQ IDNO:244.

In some embodiments, the engineered HHDH polypeptide (as compared to areference HHDH polypeptide of SEQ ID NO:244) is capable of increasedrate of converting 2-chloro-cyclohexanol to cyclohexene oxide, as shownin Scheme 11.

In some embodiments, the present disclosure provides an engineered HHDHpolypeptide that is capable of at least 1.5-fold more activity than thepolypeptide of SEQ ID NO:244 in the conversion of 2-chloro-cyclohexanolto cyclohexene oxide (Scheme 11). In some embodiments, the recombinantHHDH comprises an amino acid sequence having one or more residuedifferences (as compared to SEQ ID NO: 244) selected from: X84 is L, I,M, or F; X134 is T or V; X134 is T, X142 is L, and X245 is V. Exemplarypolypeptides with this property include, but are not limited to,polypeptides comprising amino acid sequences corresponding to SEQ ID NO:62, 220, 228, 250, 298, 302, or 304.

In some embodiments, the engineered HHDH are capable of converting anon-terminal epoxide of cis or trans 2,3-epoxybutane that is improvedover the reference polypeptide of SEQ ID NO:244. In some embodiments, inthe conversion of cis 2,3 epoxybutane, the improvement in activity areassociated with the following residue differences or groups of residuedifferences (as compared to SEQ ID NO: 244): N176M; R20C/W86H/G137W;A134V; A134L; N176H; Y1871; V84I; 163V/N176F; L142C; A82F; F12M/T671;A82Y; W86F; A82L; F12M; L142M; A134C; and D182S. Exemplary engineeredHHDH polypeptides with this improved activity comprise an amino acidsequence corresponding to SEQ ID NO: 30, 64, 66, 68, 178, 182, 186, 210,212, 214, 220, 226, 228, 238, 242, or 246. In some embodiments, theengineered HHDH has at least 1.5-fold greater activity on cis 2,3epoxybutane as compared to SEQ ID NO:244 and comprises an amino acidsequence corresponding to the sequence of SEQ ID NO: 64, 68, 220, 226,or 228.

In some embodiments, wherein the non-terminal epoxide istrans-2,3-epoxybutane, the improvements in activity are associated withthe following residue differences or groups of residue differences (ascompared to SEQ ID NO: 244): A134V; R20C/W86H/G137W; A134L; V84I; W86F;Y1871; L142M; A82Y; A82W; A82F; A134C; L178V; L142C; A82M; L178M; L178V;A82L; F12M; N176M; L142G; A82S; A82K; A82I; 181L; Y186F; A134F; A82N;A82G; A82T; N176H; V84C; L1421; A134M; and P175C. Exemplary engineeredHHDH polypeptides having this activity comprise an amino acid sequencecorresponding to SEQ ID NO: 30, 58, 64, 68, 96, 178, 182, 184, 186, 202,204, 206, 210, 212, 214, 216, 218, 220, 226, 228, 230, 232, 234, 236,238, 242, or 254.

In some embodiments, the engineered HHDH has at least 1.5 fold greateractivity on trans 2,3 epoxybutane as compared to SEQ ID NO:244 andcomprises an amino acid sequence corresponding to the sequence of SEQ IDNO: 96, 178, 186, 206, 210, 212, 214, 218, 220, 226, 228, 242, or 254.

In some embodiments, the engineered HHDH has at least 3 fold greateractivity on trans 2,3 epoxybutane as compared to SEQ ID NO:244 andcomprises an amino acid sequence of SEQ ID NO:212 or 226.

In some embodiments, the engineered HHDH has at least 10 fold greateractivity on trans 2,3 epoxybutane as compared to SEQ ID NO:244 andcomprises an amino acid sequence of SEQ ID NO:228.

Further provided herein are engineered HHDH polypeptides that haveincreased refractoriness to inhibition by a halide ion as compared to areference HHDH polypeptide of SEQ ID NO:4 or SEQ ID NO:244. In someembodiments, the halide ion is selected from Br⁻, Cl⁻, or I. In someembodiments, the HHDH polypeptides have at least about 1.5-fold, 2-fold,5-fold, or at least about 10-fold increase in refractoriness toinhibition by halide ions than SEQ ID NO: 244. In some embodiments, theengineered HHDH polypeptides having increased refractoriness toinhibition by halide ions can comprise an amino acid sequence with oneor more of the following residue differences (as compared to SEQ ID NO:244): X114 is V, and X178 is C; X167 is H, and X178 is V; X139 is M,X176 is D, and X243 is S; X139S and X249V; X187 is S; X178 is V; X12 isY or G; X139 is M, X176 is T, X223 is L, and X243 is L; X139 is M andX176 is S; or X139 is M and X176 is T. Exemplary polypeptides withhaving increased refractoriness to inhibition by halide ions include,but are not limited to, a polypeptide comprising an amino acid sequencecorresponding to SEQ ID NO: 28, 90, 94, 114, 118, 148, 172, 254, 256,260, or 268.

In some embodiments the recombinant HHDH has at least a 5-fold increasein K_(i) for halide ion as compared to the K_(i) for SEQ ID NO: 4 or SEQID NO: 244. In some embodiments, the recombinant HHDH polypeptides havea K_(i) for chloride of at least about 10 mM. In some embodiments, therecombinant HHDH polypeptides are capable of maintaining a higher rateof catalysis of haloalcohol ring closure and ring opening of epoxides inthe presence of 100 mM chloride as compared to the polypeptide of SEQ IDNO: 4 or SEQ ID NO: 244.

In some embodiments, the present disclosure provides engineered HHDHpolypeptides capable of using formate as a nucleophile in catalyzing theenantioselective ring opening reaction of an α,α-disubstituted styreneoxide of compound of formula (X) as shown below in Scheme 12.

In some embodiments, the engineered HHDH polypeptides capable ofcatalyzing enantioselective ring opening using formate as in Scheme 12have amino acid sequences comprising at least the following residuedifferences (as compared to SEQ ID NO: 244): X81E, M, or W; X82C, or S;X84I; X1341; X142G, or N; and X176D. In some embodiments, the engineeredHHDH polypeptide capable of catalyzing enantioselective ring openingusing formate as in Scheme 12 comprise an amino acid sequence of any oneof SEQ ID NO: 176, 184, 194, 196, 198, 220, 222, 230, or 240.

In some embodiments, the engineered HHDH polypeptides has an increasedrate for converting the epoxide substrate of formula (I) to the alcoholproduct of formula (II), infra, as compared to the HHDH polypeptide ofSEQ ID NO:244. In some embodiments, the epoxide substrate of formula (I)is selected from: styrene oxide (1a); para-nitrostyrene oxide (1b);benzylethylene oxide (1c); 1,2-epoxybutane (2a); and 1,2 epoxyhexane(2b) (as illustrated in the structures below).

Table 2 below provides a list of exemplary engineered HHDH polypeptidesand corresponding polynucleotides with the altered or improved enzymeproperties described herein. The odd numbered SEQ IDs refer to thepolynucleotide sequences while the even numbered SEQ ID NOs refer to theamino acid sequences. The column listing the residue differences (i.e.,mutations) is with respect to the engineered HHDH of SEQ ID NO: 244,unless indicated otherwise. The amino acid sequence of SEQ ID NO: 4 isthe wild-type sequence HheC of A. radiobacter (accession:>gi|15213643|gb|AAK92099.1|AF397296_(—)1 halohydrin dehalogenase) withtwo amino acid residue changes: C153S and W249F (relative to wild-typeHheC as reference).

TABLE 2 SEQ ID NO: Residue Differences (nt/aa) (relative to SEQ ID NO:244) 1/2 L142I; V245A 3/4 H37Q, Q38K, I52K, L70Y, H72Q, A82F, P83A,V84P, W86F, R87Q, D99G, T100A, K107R, A134T, S146T, A154T, A174G, E181E,Y186F, S189T, S195N, R203K, T222A, V245M, W249F 5/6 W249H 7/8 D39G,A60T, S68G, H72Q, A110S, T222A, A240T  9/10 K10R, A60T, P83A, K215R,T222A, W249R 11/12 G137W, M252I 13/14 L178V, W238L 15/16 V75I, W238L17/18 K91R, S189T, P196L, A240T 19/20 A4S, K91R, S189T, P196L, K215E,A240T 21/22 H72Q, A240T 23/24 H72R, W139Y, W249R 25/26 F12S, A69V, A116V27/28 F12G 29/30 F12M 31/32 W139I 33/34 W139L 35/36 W139V 37/38 S2G,W139T 39/40 W139S 41/42 W139N 43/44 P175M 45/46 P175L 47/48 P175V 49/50P175G 51/52 P175I 53/54 A134T, P175L 55/56 P175S 57/58 P175C 59/60 P175N61/62 A134T 63/64 N176H 65/66 I63V, N176F 67/68 N176M 69/70 N176Q 71/72Y177S 73/74 K10N 75/76 K10R, Y177N 77/78 Y177T 79/80 Y177L 81/82 A82T83/84 S34G 85/86 I128V, L178C 87/88 L178T 89/90 N167H, L178V 91/92 Y187G93/94 Y187S 95/96 Y187I 97/98 Y187H  99/100 W139I, W249G 101/102 W139L,A150V, W249I 103/104 W139L, W249* (sequence truncated at X248) 105/106W139P, W249A 107/108 W139G, W249S 109/110 Y186H 111/112 D182S 113/114F12Y 115/116 N113S, W139T, N176S 117/118 W139M, N176T, F223L, F243L119/120 F243L 121/122 W139M, F243S 123/124 N176S, F243L 125/126 W139T,F243L 127/128 W139S, N176D 129/130 E85V, F243S 131/132 D99N, W139M133/134 T131A, W139T, N176D 135/136 W139S, F243S 137/138 W139T, N176S,F243S 139/140 K121R, N176T, F243S 141/142 N176T 143/144 W139S, N176S,F243S 145/146 W139S, N176S 147/148 W139M, N176T 149/150 L51P, W139M,N176T, A200T, F243S 151/152 K91E, N176S, L232S 153/154 W139M, N176S,F243L 155/156 W139M, N176S, H201R, K204R 157/158 S146P, N176S, F243S159/160 N176D, F243S 161/162 W139T, N176D, F243S 163/164 W139M, N176D165/166 W139T, S180T, F243S 167/168 W139S, N176D, F243S 169/170 W139M,N176D, Y186H, F243L 171/172 W139M, N176D, F243S 173/174 I81F 175/176A82C 177/178 A82F 179/180 A82G 181/182 A82L 183/184 A82S 185/186 A82Y187/188 P83C 189/190 P83E 191/192 W86A 193/194 I81E 195/196 I81M 197/198I81W 199/200 I81Y 201/202 A82I 203/204 A82K 205/206 A82M 207/208 V84D209/210 W86F 211/212 R20C, W86H, G137W 213/214 A134C 215/216 A82N217/218 A82W 219/220 V84I 221/222 A134I 223/224 A134K 225/226 A134L227/228 A134V 229/230 L142G 231/232 A134F 233/234 A134M 235/236 L142I237/238 L142M 239/240 L142N 241/242 L142C 243/244 N/A 245/246 F12M;T67I; 247/248 P175L; T222A; 249/250 A134T 251/252 L178C; 253/254 L178V255/256 A114V; L178C; 257/258 Y186F 259/260 W139S; W249V; 261/262 W139T;W249H; 263/264 W139M; F223V; W249P; 265/266 W139T; 267/268 W139M; N176S;269/270 W139S; N176T; 271/272 G25C; W139M; N176T; 273/274 D80V; W139S;275/276 L62V; W139M; E197G; 277/278 E33G; W139T; N176S; 279/280 W139T;N176D; 281/282 T3S; W139M; N176S; 283/284 W139T; N176S; 285/286 W139M;287/288 E95G; W139M; F243S; 289/290 E85D; W139M; I168V; 291/292 W139T;H201R; 293/294 V112A; W139S; 295/296 V84C 297/298 V84F; 299/300 I81L301/302 V84L; 303/304 V84M;

In some embodiments, the engineered HHDH polypeptides comprise an aminoacid sequence that is at least about 60%, 65%, 70%, 71%, 72%, 73%, 74%75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85% 86% 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to areference sequence based on SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90,92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120,122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148,150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176,178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204,206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232,234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260,262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288,290, 292, 294, 296, 298, 300, 302, and 304 with the proviso that theengineered HHDH amino acid sequence has at least any one of the set ofthe specified residue differences (i.e., mutations) contained in any oneof the polypeptide sequences listed in Table 2. In some embodiments, theHHDH polypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7,1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24,1-25, 1-30, 1-35, 1-40, 1-45, or 1-50 residue differences at other aminoacid residue positions as compared to the reference sequence. In someembodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, 40, 45, or 50residue differences at other amino acid residues. In some embodiments,the differences comprise conservative mutations.

As noted above, the halohydrin dehalogenases of the disclosure can bedescribed in reference to the amino acid sequence of a naturallyoccurring HHDH HheC of A. radiobacter, the engineered HheC double-mutant(SEQ ID NO: 4), or another engineered HHDH (e.g., SEQ ID NO: 244). Assuch, the amino acid residue position is determined in the halohydrindehalogenases beginning from the initiating methionine (M) residue(i.e., M represents residue position 1), although it will be understoodby the skilled artisan that this initiating methionine residue may beremoved by biological processing machinery, such as in a host cell or invitro translation system, to generate a mature protein lacking theinitiating methionine residue. The amino acid residue position at whicha particular amino acid or amino acid change is present in an amino acidsequence is sometimes describe herein in terms “Xn”, or “position n”,where n refers to the residue position. Furthermore, the halohydrindehalogenases of the disclosure may be described relative to a referencepolypeptide sequence. For example, one reference sequence is that of anengineered double-mutant of A. radiobacter HheC which comprises theamino acid sequence of SEQ ID NO: 4. Another reference sequence is thatof SEQ ID NO: 244 which is the amino acid sequence of SEQ ID NO:730disclosed in U.S. Ser. No. 11/266,747, published as US 2006/0099700 A1,which is hereby incorporated by reference in its entirety.

As described in the foregoing, the halohydrin dehalogenase polypeptidesherein can have a number of modifications to the reference sequence(e.g., naturally occurring polypeptide or an engineered polypeptide) toresult in the improved halohydrin dehalogenase property. As used herein,“modifications” include amino acid substitutions, deletions, andinsertions. Any one or a combination of modifications can be introducedinto the naturally occurring or engineered polypeptide to generateengineered enzymes. In such embodiments, the number of modifications tothe amino acid sequence can comprise one or more amino acids, 2 or moreamino acids, 3 or more amino acids, 4 or more amino acids, 5 or moreamino acids, 6 or more amino acids, 8 or more amino acids, 10 or moreamino acids, 15 or more amino acids, or 20 or more amino acids, up to10% of the total number of amino acids, up to 15% of the total number ofamino acids, up to 20% of the total number of amino acids, or up to 30%of the total number of amino acids of the reference polypeptidesequence. In some embodiments, the number of modifications to thenaturally occurring polypeptide or an engineered polypeptide thatproduces an improved halohydrin dehalogenase property may comprise fromabout 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14,1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35 or about 1-40modifications of the reference sequence. In some embodiments, the numberof modifications can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15,16, 18, 20, 22, 24, 26, 30, 35 or about 40 amino acid residues. Themodifications can comprise insertions, deletions, substitutions, orcombinations thereof.

In some embodiments, the modifications comprise amino acid substitutionsto the reference sequence. Substitutions that can produce an improvedhalohydrin dehalogenase property may be at one or more amino acids, 2 ormore amino acids, 3 or more amino acids, 4 or more amino acids, 5 ormore amino acids, 6 or more amino acids, 8 or more amino acids, 10 ormore amino acids, 15 or more amino acids, or 20 or more amino acids, upto 10% of the total number of amino acids, up to 15% of the total numberof amino acids, up to 20% of the total number of amino acids, or up to30% of the total number of amino acids of the reference enzyme sequence.In some embodiments, the number of substitutions to the naturallyoccurring polypeptide or an engineered polypeptide that produces animproved halohydrin dehalogenase property can comprise from about 1-2,1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16,1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35 or about 1-40 amino acidsubstitutions of the reference sequence. In some embodiments, the numberof substitutions can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15,16, 18, 20, 22, 24, 26, 30, 35 or about 40 amino acid residues.

As will be appreciated by those of skill in the art, some of theabove-defined categories of amino acid residues, unless otherwisespecified, are not mutually exclusive. Thus, amino acids having sidechains exhibiting two or more physico-chemical properties can beincluded in multiple categories. The appropriate classification of anyamino acid or residue will be apparent to those of skill in the art,especially in light of the detailed disclosure provided herein.

In some embodiments, the improved engineered halohydrin dehalogenaseenzymes comprise deletions of the naturally occurring halohydrindehalogenase polypeptides or deletions of other engineered halohydrindehalogenase polypeptides. In some embodiments, each of the improvedengineered halohydrin dehalogenase enzymes described herein can comprisedeletions of the polypeptides described herein. Thus, for each and everyembodiment of the halohydrin dehalogenase polypeptides of thedisclosure, the deletions can comprise one or more amino acids, 2 ormore amino acids, 3 or more amino acids, 4 or more amino acids, 5 ormore amino acids, 6 or more amino acids, 8 or more amino acids, 10 ormore amino acids, 15 or more amino acids, or 20 or more amino acids, upto 10% of the total number of amino acids, up to 15% of the total numberof amino acids, up to 20% of the total number of amino acids, or up to30% of the total number of amino acids of the halohydrin dehalogenasepolypeptides, as long as the functional activity of the halohydrindehalogenase activity is maintained. In some embodiments, the deletionscan comprise, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12,1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-25, 1-30, 1-35 or about 1-40amino acid residues. In some embodiments, the number of deletions can be1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26,30, 35 or about 40 amino acids. In some embodiments, the deletions cancomprise deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 18, or 20 amino acid residues.

In some embodiments, the present disclosure provides an engineered HHDHpolypeptide having a C-terminal 6 amino deletion relative to thereference polypeptides of SEQ ID NO: 4 or 244. In such an embodiment,the resulting engineered HHDH polypeptide is only 248 amino acidresidues (rather than 254) in length. Accordingly, in some embodiments,the present disclosure provides engineered HHDH polypeptides withimproved enzyme properties relative to reference polypeptides of SEQ IDNO: 4 or 244, wherein the amino acid sequence consists of an amino acidsequence from position X1 to X248, or from position X2 to X248, of anyone of any one of the polypeptides of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144,146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172,174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200,202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228,230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256,258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284,286, 288, 290, 292, 294, 296, 298, 300, 302, and 304. In one embodiment,the C-terminal truncated engineered HHDH polypeptide comprises the aminoacid sequence of SEQ ID NO: 104.

As described herein, the engineered HHDH polypeptides of the disclosurecan be in the form of fusion polypeptides in which the halohydrindehalogenase polypeptides are fused to other polypeptides, such as, byway of example and not limitation, antibody tags (e.g., myc epitope),purifications sequences (e.g., His tags), and cell localization signals(e.g., secretion signals). Thus, the halohydrin dehalogenasepolypeptides can be used with or without fusions to other polypeptides.

Accordingly, in some embodiments, the engineered HHDH polypeptides canfurther comprises a fusion at the carboxy terminus of a contiguoussegment of about 1 to about 20 amino acid residues to a polypeptide ofany one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126,128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154,156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182,184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210,212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238,240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266,268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294,296, 298, 300, 302, and 304. In some embodiments, any of the engineeredHHDH polypeptides can be modified with a one residue extension atposition X254 of an asparagine residue (N). Thus, in some embodiments,any of the engineered HHDH polypeptides sequences disclosed herein maycomprise at least a position X255 having a N residue (i.e., X255N). Ithas been observed that an engineered HHDH polypeptide comprising theamino acid sequence of SEQ ID NO: 126 can be modified with a C-terminalasparagine (X255N) and is capable of improved enzyme properties ofincreased activity and enantioselectivity relative to SEQ ID NO: 244. Itis contemplated that this or other C-terminal fusions to the disclosedengineered polypeptides can result in similarly improved properties.

The polypeptides described herein are not restricted to the geneticallyencoded amino acids. In addition to the genetically encoded amino acids,the polypeptides described herein may be comprised, either in whole orin part, of naturally-occurring and/or synthetic non-encoded aminoacids. Certain commonly encountered non-encoded amino acids of which thepolypeptides described herein may be comprised include, but are notlimited to: the D-stereomers of the genetically-encoded amino acids;2,3-diaminopropionic acid (Dpr); α-aminoisobutyric acid (Aib);ε-aminohexanoic acid (Aha); δ-aminovaleric acid (Ava); N-methylglycineor sarcosine (MeGly or Sar); ornithine (Orn); citrulline (Cit);t-butylalanine (Bua); t-butylglycine (Bug); N-methylisoleucine (MeIle);phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (Nle);naphthylalanine (NaI); 2-chlorophenylalanine (Ocf);3-chlorophenylalanine (Mcf); 4-chlorophenylalanine (Pcf);2-fluorophenylalanine (Off); 3-fluorophenylalanine (Mff);4-fluorophenylalanine (Pff); 2-bromophenylalanine (Obf);3-bromophenylalanine (Mbf); 4-bromophenylalanine (Pbf);2-methylphenylalanine (Omf); 3-methylphenylalanine (Mmf);4-methylphenylalanine (Pmf); 2-nitrophenylalanine (Onf);3-nitrophenylalanine (Mnf); 4-nitrophenylalanine (Pnf);2-cyanophenylalanine (Ocf); 3-cyanophenylalanine (Mcf);4-cyanophenylalanine (Pcf); 2-trifluoromethylphenylalanine (Otf);3-trifluoromethylphenylalanine (Mtf); 4-trifluoromethylphenylalanine(Ptf); 4-aminophenylalanine (Paf); 4-iodophenylalanine (Pif);4-aminomethylphenylalanine (Pamf); 2,4-dichlorophenylalanine (Opef);3,4-dichlorophenylalanine (Mpcf); 2,4-difluorophenylalanine (Opff);3,4-difluorophenylalanine (Mpff); pyrid-2-ylalanine (2pAla);pyrid-3-ylalanine (3pAla); pyrid-4-ylalanine (4pAla); naphth-1-ylalanine(1nAla); naphth-2-ylalanine (2nAla); thiazolylalanine (taAla);benzothienylalanine (bAla); thienylalanine (tAla); furylalanine (fAla);homophenylalanine (hPhe); homotyrosine (hTyr); homotryptophan (hTrp);pentafluorophenylalanine (5ff); styrylkalanine (sAla); authrylalanine(aAla); 3,3-diphenylalanine (Dfa); 3-amino-5-phenypentanoic acid (Afp);penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(Tic); β-2-thienylalanine (Thi); methionine sulfoxide (Mso);N(w)-nitroarginine (nArg); homolysine (hLys);phosphonomethylphenylalanine (pmPhe); phosphoserine (pSer);phosphothreonine (pThr); homoaspartic acid (hAsp); homoglutanic acid(hGlu); 1-aminocyclopent-(2 or 3)-ene-4 carboxylic acid; pipecolic acid(PA), azetidine-3-carboxylic acid (ACA);1-aminocyclopentane-3-carboxylic acid; allylglycine (aOly);propargylglycine (pgGly); homoalanine (hAla); norvaline (nVal);homoleucine (hLeu), homovaline (hVal); homoisolencine (hIle);homoarginine (hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric acid(Dbu); 2,3-diaminobutyric acid (Dab); N-methylvaline (MeVal);homocysteine (hCys); homoserine (hSer); hydroxyproline (Hyp) andhomoproline (hPro). Additional non-encoded amino acids of which thepolypeptides described herein may be comprised will be apparent to thoseof skill in the art (see, e.g., the various amino acids provided inFasman, 1989, CRC Practical Handbook of Biochemistry and MolecularBiology, CRC Press, Boca Raton, Fla., at pp. 3-70 and the referencescited therein, all of which are incorporated by reference). These aminoacids may be in either the L- or D-configuration.

Those of skill in the art will recognize that amino acids or residuesbearing side chain protecting groups may also comprise the polypeptidesdescribed herein. Non-limiting examples of such protected amino acids,which in this case belong to the aromatic category, include (protectinggroups listed in parentheses), but are not limited to: Arg(tos),Cys(methylbenzyl), Cys (nitropyridinesulfenyl), Glu(δ-benzylester),Gln(xanthyl), Asn(N-δ-xanthyl), His(bom), His(benzyl), His(tos),Lys(fmoc), Lys(tos), Ser(O-benzyl), Thr (O-benzyl) and Tyr(O-benzyl).

Non-encoding amino acids that are conformationally constrained of whichthe polypeptides described herein may be composed include, but are notlimited to, N-methyl amino acids (L-configuration); 1-aminocyclopent-(2or 3)-ene-4-carboxylic acid; pipecolic acid; azetidine-3-carboxylicacid; homoproline (hPro); and 1-aminocyclopentane-3-carboxylic acid.

As described above the various modifications introduced into thenaturally occurring polypeptide to generate an engineered halohydrindehalogenase enzyme can be targeted to a specific property of theenzyme.

1.4 Polynucleotides Encoding Engineered Halohydrin Dehalogenases

In another aspect, the present disclosure provides polynucleotidesencoding the engineered halohydrin dehalogenase enzymes. Thepolynucleotides may be operatively linked to one or more heterologousregulatory sequences that control gene expression to create arecombinant polynucleotide capable of expressing the polypeptide.Expression constructs containing a heterologous polynucleotide encodingthe engineered halohydrin dehalogenase can be introduced intoappropriate host cells to express the corresponding halohydrindehalogenase polypeptide.

Because of the knowledge of the codons corresponding to the variousamino acids, availability of a protein sequence provides a descriptionof all the polynucleotides capable of encoding the subject. Thedegeneracy of the genetic code, where the same amino acids are encodedby alternative or synonymous codons allows an extremely large number ofnucleic acids to be made, all of which encode the improved halohydrindehalogenase enzymes disclosed herein. Thus, having identified aparticular amino acid sequence, those skilled in the art could make anynumber of different nucleic acids by simply modifying the sequence ofone or more codons in a way which does not change the amino acidsequence of the protein. In this regard, the present disclosurespecifically contemplates each and every possible variation ofpolynucleotides that could be made by selecting combinations based onthe possible codon choices, and all such variations are to be consideredspecifically disclosed for any polypeptide disclosed herein, includingthe amino acid sequences presented in Table 2 and the accompanyingSequence Listing.

In various embodiments, the codons are preferably selected to fit thehost cell in which the protein is being produced. For example, preferredcodons used in bacteria are used to express the gene in bacteria;preferred codons used in yeast are used for expression in yeast; andpreferred codons used in mammals are used for expression in mammaliancells.

In certain embodiments, all codons need not be replaced to optimize thecodon usage of the halohydrin dehalogenases since the natural sequencewill comprise preferred codons and because use of preferred codons maynot be required for all amino acid residues. Consequently, codonoptimized polynucleotides encoding the halohydrin dehalogenase enzymesmay contain preferred codons at about 40%, 50%, 60%, 70%, 80%, orgreater than 90% of codon positions of the full length coding region.

In some embodiments, the polynucleotide comprises a nucleotide sequenceencoding a halohydrin dehalogenase polypeptide with an amino acidsequence that has at least about 60%, 65%, 70%, 71%, 72%, 73%, 74% 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85% 86% 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity toa reference engineered halohydrin dehalogenase polypeptides describedherein. Accordingly, in some embodiments, the polynucleotide encodes anamino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical tothe sequence of SEQ ID NO: 244 and differs from SEQ ID NO244 at one ormore residue positions selected from: X34; X51; X81; X85; X110; X131;X167; X232; X242; X248, and X254. In some embodiments, thepolynucleotide encodes an amino acid sequence corresponding to SEQ IDNO: 2, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74,76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164,166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192,194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220,222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248,250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276,278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, and304.

In some embodiments, the polynucleotide comprises a nucleotide sequenceencoding a halohydrin dehalogenase polypeptide with an amino acidsequence that has at least about 60%, 65%, 70%, 71%, 72%, 73%, 74% 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85% 86% 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity tothe polypeptide comprising an amino acid corresponding to SEQ ID NO: 2,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164,166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192,194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220,222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248,250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276,278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, and304.

In some embodiments, the polynucleotides encoding the halohydrindehalogenases are selected from SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89,91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119,121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147,149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175,177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203,205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231,233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259,261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287,289, 291, 293, 295, 297, 299, 301, and 303.

In some embodiments, the polynucleotides are capable of hybridizingunder highly stringent conditions to a polynucleotide comprising SEQ IDNO: 1, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73,75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107,109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135,137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163,165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191,193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219,221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247,249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275,277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, and303, where the highly stringently hybridizing polynucleotides encode ahalohydrin dehalogenase capable of stereoselectively converting thesubstrate of formula (I) to the product of formula (II), orstereoselectively converting the substrate of formula (I) to the productof formula (III), or stereoselectively converting a substrate of formula(IV) to the product of formula (V).

In some embodiments, the polynucleotides encode the polypeptidesdescribed herein but have about 80% or more sequence identity, about80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% or more sequence identity at the nucleotide level to areference polynucleotide encoding the engineered halohydrindehalogenase. In some embodiments, the reference polynucleotide isselected from SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63,65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99,101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127,129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155,157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183,185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211,213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239,241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267,269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295,297, 299, 301, and 303.

An isolated polynucleotide encoding an improved halohydrin dehalogenasepolypeptide may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the isolatedpolynucleotide prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying polynucleotides and nucleic acid sequences utilizingrecombinant DNA methods are well known in the art. Guidance is providedin Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3^(rd)Ed., Cold Spring Harbor Laboratory Press; and Current Protocols inMolecular Biology, Ausubel. F. ed., Greene Pub. Associates, 1998,updates to 2006.

For bacterial host cells, suitable promoters for directing transcriptionof the nucleic acid constructs of the present disclosure, include thepromoters obtained from the E. coli lac operon, E. coli trp operon,bacteriophage λ, Streptomyces coelicolor agarase gene (dagA), Bacillussubtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylasegene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as the tac promoter(DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80: 21-25). Furtherpromoters are described in “Useful proteins from recombinant bacteria”in Scientific American, 1980, 242:74-94; and in Sambrook et al., supra.

For filamentous fungal host cells, suitable promoters for directing thetranscription of the nucleic acid constructs of the present disclosureinclude promoters obtained from the genes for Aspergillus oryzae TAKAamylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, andFusarium oxysporum trypsin-like protease (WO 96/00787), as well as theNA2-tpi promoter (a hybrid of the promoters from the genes forAspergillus niger neutral alpha-amylase and Aspergillus oryzae thosephosphate isomerase), and mutant, truncated, and hybrid promotersthereof.

In a yeast host, useful promoters can be from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), andSaccharomyces cerevisiae 3-phosphoglycerate kinase. Other usefulpromoters for yeast host cells are described by Romanos et al., 1992,Yeast 8:423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

For example, exemplary transcription terminators for filamentous fungalhost cells can be obtained from the genes for Aspergillus oryzae TAKAamylase, Aspergillus niger glucoamylase, Aspergillus nidulansanthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusariumoxysporum trypsin-like protease.

Exemplary terminators for yeast host cells can be obtained from thegenes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C(CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA that is important for translation by thehost cell. The leader sequence is operably linked to the 5′ terminus ofthe nucleic acid sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used. Exemplaryleaders for filamentous fungal host cells are obtained from the genesfor Aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase. Suitable leaders for yeast host cells are obtainedfrom the genes for Saccharomyces cerevisiae enolase (ENO-1),Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomycescerevisiae alpha-factor, and Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleic acid sequence andwhich, when transcribed, is recognized by the host cell as a signal toadd polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention. Exemplary polyadenylation sequences forfilamentous fungal host cells can be from the genes for Aspergillusoryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillusnidulans anthranilate synthase, Fusarium oxysporum trypsin-likeprotease, and Aspergillus niger alpha-glucosidase. Usefulpolyadenylation sequences for yeast host cells are described by Guo andSherman, 1995, Mol Cell Bio 15:5983-5990.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion that encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region thatis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region.

Alternatively, the foreign signal peptide coding region may simplyreplace the natural signal peptide coding region in order to enhancesecretion of the polypeptide. However, any signal peptide coding regionwhich directs the expressed polypeptide into the secretory pathway of ahost cell of choice may be used in the present invention.

Effective signal peptide coding regions for bacterial host cells are thesignal peptide coding regions obtained from the genes for Bacillus NC1B11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase,Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiol Rev 57: 109-137.

Effective signal peptide coding regions for filamentous fungal hostcells can be the signal peptide coding regions obtained from the genesfor Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, and Humicola lanuginosa lipase.

Useful signal peptides for yeast host cells can be from the genes forSaccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalactase (WO 95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences, which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. In prokaryotic host cells, suitable regulatory sequencesinclude the lac, tac, and trp operator systems. In yeast host cells,suitable regulatory systems include, as examples, the ADH2 system orGAL1 system. In filamentous fungi, suitable regulatory sequences includethe TAKA alpha-amylase promoter, Aspergillus niger glucoamylasepromoter, and Aspergillus oryzae glucoamylase promoter.

Other examples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene, which is amplified in the presence of methotrexate, andthe metallothionein genes, which are amplified with heavy metals. Inthese cases, the nucleic acid sequence encoding the HHDH polypeptide ofthe present invention would be operably linked with the regulatorysequence.

Thus, in another embodiment, the present disclosure is also directed toa recombinant expression vector comprising a polynucleotide encoding anengineered halohydrin dehalogenase polypeptide or a variant thereof, andone or more expression regulating regions such as a promoter and aterminator, a replication origin, etc., depending on the type of hostsinto which they are to be introduced. The various nucleic acid andcontrol sequences described above may be joined together to produce arecombinant expression vector which may include one or more convenientrestriction sites to allow for insertion or substitution of the nucleicacid sequence encoding the polypeptide at such sites. Alternatively, thenucleic acid sequence of the present disclosure may be expressed byinserting the nucleic acid sequence or a nucleic acid constructcomprising the sequence into an appropriate vector for expression. Increating the expression vector, the coding sequence is located in thevector so that the coding sequence is operably linked with theappropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus), which can be conveniently subjected to recombinant DNAprocedures and can bring about the expression of the polynucleotidesequence. The choice of the vector will typically depend on thecompatibility of the vector with the host cell into which the vector isto be introduced. The vectors may be linear or closed circular plasmids.

The expression vector may be an autonomously replicating vector, i.e., avector that exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The expression vector of the present invention preferably contains oneor more selectable markers, which permit easy selection of transformedcells. A selectable marker is a gene the product of which provides forbiocide or viral resistance, resistance to heavy metals, prototrophy toauxotrophs, and the like. Examples of bacterial selectable markers arethe dal genes from Bacillus subtilis or Bacillus licheniformis, ormarkers, which confer antibiotic resistance such as ampicillin,kanamycin, chloramphenicol (Example 1) or tetracycline resistance.Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3,TRP1, and URA3.

Selectable markers for use in a filamentous fungal host cell include,but are not limited to, amdS (acetamidase), argB (ornithinecarbamoyltransferase), bar (phosphinothricin acetyltransferase), hph(hygromycin phosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Embodiments for use in an Aspergillus cell include the amdS and pyrGgenes of Aspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The expression vectors of the present invention preferably contain anelement(s) that permits integration of the vector into the host cell'sgenome or autonomous replication of the vector in the cell independentof the genome. For integration into the host cell genome, the vector mayrely on the nucleic acid sequence encoding the polypeptide or any otherelement of the vector for integration of the vector into the genome byhomologous or nonhomologous recombination.

Alternatively, the expression vector may contain additional nucleic acidsequences for directing integration by homologous recombination into thegenome of the host cell. The additional nucleic acid sequences enablethe vector to be integrated into the host cell genome at a preciselocation(s) in the chromosome(s). To increase the likelihood ofintegration at a precise location, the integrational elements shouldpreferably contain a sufficient number of nucleic acids, such as 100 to10,000 base pairs, preferably 400 to 10,000 base pairs, and mostpreferably 800 to 10,000 base pairs, which are highly homologous withthe corresponding target sequence to enhance the probability ofhomologous recombination. The integrational elements may be any sequencethat is homologous with the target sequence in the genome of the hostcell. Furthermore, the integrational elements may be non-encoding orencoding nucleic acid sequences. On the other hand, the vector may beintegrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of bacterial origins of replication are P15Aori (as shown in the plasmid of FIG. 5) or the origins of replication ofplasmids pBR322, pUC19, pACYC177 (which plasmid has the P15A ori), orpACYC184 permitting replication in E. coli, and pUB110, pE194, pTA1060,or pAMλ1 permitting replication in Bacillus. Examples of origins ofreplication for use in a yeast host cell are the 2 micron origin ofreplication, ARS1, ARS4, the combination of ARS1 and CEN3, and thecombination of ARS4 and CEN6. The origin of replication may be onehaving a mutation which makes it's functioning temperature-sensitive inthe host cell (see, e.g., Ehrlich, 1978, Proc Natl Acad. Sci. USA75:1433).

More than one copy of a nucleic acid sequence of the present inventionmay be inserted into the host cell to increase production of the geneproduct. An increase in the copy number of the nucleic acid sequence canbe obtained by integrating at least one additional copy of the sequenceinto the host cell genome or by including an amplifiable selectablemarker gene with the nucleic acid sequence where cells containingamplified copies of the selectable marker gene, and thereby additionalcopies of the nucleic acid sequence, can be selected for by cultivatingthe cells in the presence of the appropriate selectable agent.

Many of the expression vectors for use in the present invention arecommercially available. Suitable commercial expression vectors includep3×FLAG™™ expression vectors from Sigma-Aldrich Chemicals, St. LouisMo., which includes a CMV promoter and hGH polyadenylation site forexpression in mammalian host cells and a pBR322 origin of replicationand ampicillin resistance markers for amplification in E. coli. Othersuitable expression vectors are pBluescriptII SK(−) and pBK-CMV, whichare commercially available from Stratagene, LaJolla Calif., and plasmidswhich are derived from pBR322 (Gibco BRL), pUC (Gibco BRL), pREP4, pCEP4(Invitrogen) or pPoly (Lathe et al., 1987, Gene 57:193-201).

1.5 Host Cells for Expression of Halohydrin Dehalogenase Polypeptides

In another aspect, the present disclosure provides a host cellcomprising a polynucleotide encoding an improved halohydrin dehalogenasepolypeptide of the present disclosure, the polynucleotide beingoperatively linked to one or more control sequences for expression ofthe halohydrin dehalogenase enzyme in the host cell. Host cells for usein expressing the HHDH polypeptides encoded by the expression vectors ofthe present invention are well known in the art and include but are notlimited to, bacterial cells, such as E. coli, Lactobacillus kefir,Lactobacillus brevis, Streptomyces and Salmonella typhimurium cells;fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae orPichia pastoris (ATCC Accession No. 201178)); insect cells such asDrosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS,BHK, 293, and Bowes melanoma cells; and plant cells. Appropriate culturemediums and growth conditions for the above-described host cells arewell known in the art.

Polynucleotides for expression of the halohydrin dehalogenase may beintroduced into cells by various methods known in the art. Techniquesinclude among others, electroporation, biolistic particle bombardment,liposome mediated transfection, calcium chloride transfection, andprotoplast fusion. Various methods for introducing polynucleotides intocells will be apparent to the skilled artisan.

An exemplary host cell is Escherichia coli W3110. The expression vectorwas created by operatively linking a polynucleotide encoding an improvedhalohydrin dehalogenase into the plasmid pCK110900 operatively linked tothe lac promoter under control of the lad repressor. The expressionvector also contained the P15a origin of replication and thechloramphenicol resistance gene. Cells containing the subjectpolynucleotide in Escherichia coli W3110 were isolated by subjecting thecells to chloramphenicol selection. Another exemplary host cell isEscherichia coli BL21.

1.6 Methods of Generating Engineered Halohydrin DehalogenasePolypeptides

In some embodiments, to make the improved HHDH polynucleotides andpolypeptides of the present disclosure, the naturally-occurringhalohydrin dehalogenase enzyme that catalyzes the conversion reaction isobtained (or derived) from Agrobacterium radiobacter or Corynebacteriumsp. In some embodiments, the parent polynucleotide sequence is codonoptimized to enhance expression of the halohydrin dehalogenase in aspecified host cell.

The engineered halohydrin dehalogenases can be obtained by subjectingthe polynucleotide encoding the naturally occurring halohydrindehalogenase to mutagenesis and/or directed evolution methods, asdiscussed above. An exemplary directed evolution technique ismutagenesis and/or DNA shuffling as described in Stemmer, 1994, ProcNatl Acad Sci USA 91:10747-10751; WO 95/22625; WO 97/0078; WO 97/35966;WO 98/27230; WO 00/42651; WO 01/75767 and U.S. Pat. No. 6,537,746. Otherdirected evolution procedures that can be used include, among others,staggered extension process (StEP), in vitro recombination (Zhao et al.,1998, Nat. Biotechnol. 16:258-261), mutagenic PCR (Caldwell et al.,1994, PCR Methods Appl. 3:S136-S140), and cassette mutagenesis (Black etal., 1996, Proc Natl Acad Sci USA 93:3525-3529).

The clones obtained following mutagenesis treatment are screened forengineered halohydrin dehalogenases having a desired improved enzymeproperty. Clones containing a polynucleotide encoding a halohydrindehalogenase are then isolated, sequenced to identify the nucleotidesequence changes (if any), and used to express the enzyme in a hostcell.

Where the sequence of the engineered polypeptide is known, thepolynucleotides encoding the enzyme can be prepared by standardsolid-phase methods, according to known synthetic methods. In someembodiments, fragments of up to about 100 bases can be individuallysynthesized, then joined (e.g., by enzymatic or chemical ligationmethods, or polymerase mediated methods) to form any desired continuoussequence. For example, polynucleotides and oligonucleotides of theinvention can be prepared by chemical synthesis using, e.g., theclassical phosphoramidite method described by Beaucage et al., 1981, TetLett 22:1859-69, or the method described by Matthes et al., 1984, EMBOJ. 3:801-05, e.g., as it is typically practiced in automated syntheticmethods. According to the phosphoramidite method, oligonucleotides aresynthesized, e.g., in an automatic DNA synthesizer, purified, annealed,ligated and cloned in appropriate vectors. In addition, essentially anynucleic acid can be obtained from any of a variety of commercialsources, such as The Midland Certified Reagent Company, Midland, Tex.,The Great American Gene Company, Ramona, Calif., ExpressGen Inc.Chicago, Ill., Operon Technologies Inc., Alameda, Calif., and manyothers.

Engineered halohydrin dehalogenase enzymes expressed in a host cell canbe recovered from the cells and or the culture medium using any one ormore of the well known techniques for protein purification, including,among others, lysozyme treatment, sonication, high pressurehomogenization (French Press), filtration, salting-out,ultra-centrifugation, and chromatography. Suitable solutions for lysingand the high efficiency extraction of proteins from bacteria, such as E.coli, are commercially available under the trade name CelLytic B™ fromSigma-Aldrich of St. Louis Mo.

Chromatographic techniques for isolation of the halohydrin dehalogenasepolypeptide include, among others, reverse phase chromatography highperformance liquid chromatography, ion exchange chromatography, gelelectrophoresis, and affinity chromatography. Conditions for purifying aparticular enzyme will depend, in part, on factors such as net charge,hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc.,and will be apparent to those having skill in the art.

In some embodiments, affinity techniques may be used to isolate theimproved halohydrin dehalogenase enzymes. For affinity chromatographypurification, any antibody which specifically binds the halohydrindehalogenase polypeptide may be used. For the production of antibodies,various host animals, including but not limited to rabbits, mice, rats,etc., may be immunized by injection with an engineered polypeptide. Thepolypeptide may be attached to a suitable carrier, such as BSA, by meansof a side chain functional group or linkers attached to a side chainfunctional group. Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacilli Calmette Guerin) and Corynebacterium parvum.

1.7 Methods of Using Engineered Halohydrin Dehalogenase Polypeptides

In another aspect, the present disclosure provides methods of using theengineered HHDH polypeptides for enantioselective ring opening reactionswith epoxide substrates to prepare enantiospecific alcohols and/orresolve racemic epoxides. In another aspect, the present disclosureprovides methods of using the engineered HHDH polypeptides forenantioselective ring closure reactions with alcohol substrates toprepare enantiospecific epoxides and/or resolve racemic epoxides.Illustrative ring opening and ring closing reactions that can be carriedout using the engineered HHDH polypeptides of the present disclosure areprovided in the Examples below.

In some embodiments, the HHDH enzymes described herein are capable ofcatalyzing the conversion of an epoxide in the compound of structuralformula (I) (a “substrate”) to the S-alcohol product of structuralformula (II) (a “product”):

wherein, R¹ is a substituted or unsubstituted alkyl, cylcoalkyl,heteocycloalkyl, aryl, or heteroaryl; R² is H, or substituted orunsubstituted alkyl, cylcoalkyl, heteocycloalkyl, aryl, or heteroaryl;or wherein R¹ and R² forms a ring, such as a substituted orunsubstituted cycloalkyl or heterocycloalkyl; and Nuc is a suitablenucleophile, including but not limited to, Br⁻, Cl⁻, I⁻, NO₂ ⁻, N₃ ⁻,CN⁻, OCN⁻, SCN⁻, or formate (HCOO⁻). In some embodiments, R¹ is asubstituted or unsubstituted aryl, including among others, substitutedor unsubstituted phenyl. In some embodiments, the epoxide of formula (I)is styrene oxide or para-nitro styrene oxide.

In some embodiments, the halohydrin dehalogenase polypeptides of theinvention catalyze the enantioselective conversion of an epoxide in thecompound of structural formula (I) to the R-alcohol “product” of formula(III):

wherein, R¹, R² and Nuc are described above.

In some embodiments, the HHDH polypeptides of the invention can be usedfor the enantioselective conversion of an epoxide of structural formula(I) to the S-alcohol product of formula (IIIb):

wherein R¹, R², and Nuc are as described previously.

In some embodiments, the HHDH polypeptides of the invention can be usedfor the enantioselective conversion of an epoxide of structural formula(I) to the S-alcohol product of formula (VIII):

wherein R¹, R², and Nuc are as described previously.

In some embodiments, the HHDH polypeptides of the invention can be usedfor the enantioselective conversion of an epoxide of structural formula(I) to the S-alcohol product of formula (VIIIb):

wherein R¹, R², and Nuc are as described previously. In the structuresof formula (III), (Mb), (VIII) and (VIIIb), the carbon bonded to R¹ canbe the alpha carbon of the epoxide ring.

In some embodiments, the HHDH polypeptides of the inventionstereoselectively catalyze the conversion of the alcohol substrate offormula (IV), where X is halide:

to the S-epoxide product of structural formula (V).

In some embodiments, the HHDH polypeptides of the inventionstereoselectively catalyze the conversion of the alcohol substrate offormula (IV), where X is halide:

to the R-epoxide product of structural formula (IX).

Accordingly, in some embodiments, provided herein are methods forconverting the epoxide of formula (I) to the alcohol of formula (II),formula (III), formula (Mb), formula (VIII), or formula (VIIIb), whichmethod comprises contacting or incubating the epoxide with a halohydrindehalogenase polypeptide of the disclosure in the presence of anucleophile and under reaction conditions suitable for converting thesubstrate to the alcohol of structural formula (II), formula (III),formula (Mb), formula (VIII), or formula (VIIIb). In some embodiments,the nucleophile is selected from Br⁻, Cl⁻, I⁻, NO₂ ⁻, N₃ ⁻, CN⁻, OCN⁻,SCN⁻, or formate (HCOO⁻). In some embodiments, the epoxide is styreneoxide.

In some embodiments, provided herein are methods for converting thealcohol of formula (II), formula (III), or formula (IV) to the epoxideof formula (I), formula (V), or formula (IX) which method comprisescontacting or incubating the alcohol with a halohydrin dehalogenasepolypeptide of the disclosure under reaction conditions suitable forconverting the substrate to the epoxide of structural formula (I),formula (V), or formula (IX).

Any of the above-described methods of using engineered HHDH polypeptidescan be carried out using whole cells capable of expressing thepolypeptide, cell extracts, or purified HHDH enzymes. Furthermore, themethods can be carried out using a single HHDH enzyme or, alternatively,mixtures of two or more HHDH enzymes.

6. EXAMPLES

Various features and embodiments of the disclosure are illustrated inthe following representative examples, which are intended to beillustrative, and not limiting.

Example 1 Engineered HHDH Polypeptides Capable of Catalyzing RingClosure of PNSHH to PNSO with Improved Enantioselectivity

The ring closure reaction converting PNSHH (compound (VI)) to PNSO(compound (VII)) is illustrated in Scheme 13 below.

A 100 mM stock solution of compound (VI) is prepared by dissolving 24.9mg of compound (VI) in 1 mL of DMSO. The engineered HHDH enzyme stocksolution is prepared by dissolving 10 mg dry lyophilized enzyme powderin 1 mL of a 100 mM Tris-SO₄ buffer of pH 7.5. The enzyme is diluted tothe appropriate final concentration by adding 10 μL of enzyme stocksolution to 940 μL, 100 mM Tris-SO₄ buffer of pH 7.5. The reaction isthen started by adding 50 μL of compound (VI) to an end concentration of5 mM (20× dilution) and the total volume is 1 mL. Note that not everyenzyme variant is equally active and some tests should be done tooptimize the final enzyme concentration to make sure the reaction is nottoo fast, resulting in a lower e.e. of the product.

Sample preparation is done as follows. Take a sample of the reactionmixture and extract with two volumes of heptane. Analysis of the organiclayer is done on a Daicel Chiralcel AD-H column (0.46 cm×250 mm)equipped with AD-H guard column using heptane/ethanol 95:5 as eluent anda flow of 1 ml/min with detection at 216 nm. Under these conditions,baseline separation of the product compound (VII) is obtained but not ofcompound (VI). Elution order: R-product (5.3 min), S-product (5.8 min),compound (VI) (6.4 min, non-separated).

The enantioselectivity of the enzyme can be calculated from the percentconversion and the percent enantiomeric excess of the product using theformula of Chen et al., or be easily visualized by plotting % c vs. %e.e. Engineered HHDH polypeptides exhibiting improved enantioselectivityin catalyzing the ring closure reaction are listed below in Table 3.

TABLE 3 SEQ ID Residue Differences E-Value NO: (relative to SEQ ID NO:244) Selectivity Result 44 P175M; R ++++ 46 P175L; R ++++ 48 P175V; R++++ 50 P175G; R ++++ 86 I128V; L178C; R + 244 N/A R + 248 P175L; T222A;R ++++ 252 L178C; R + 256 A114V; L178C; R ++ 260 W139S; W249V; S + 262W139T; W249H; S + 264 W139M; F223V; W249P; S + 266 W139T; S + +: E-value0 to 10 ++: E-value 20 to <40 +++: E-value 40 to 100 ++++: E-value >100

Example 2 Engineered HHDH Polypeptides Capable of Catalyzing the RingOpening of Styrene Oxide with Azide to Provide 1-Azido-2-Phenylethanolwith Improved Enantioselectivity

A deep well plate with HHDH enzymes is thawed at room temperature for˜30 min and centrifuged at 4,000 rpm (3220×g) for 5 min at 4° C. to spindown any condensation.

Prepare a 200 mM styrene oxide stock solution in DMSO by dissolving 33mg/mL. To prepare a 30 mL volume of aqueous substrate buffer, add 1250μL of the 200 mM Styrene oxide stock solution to 28.75 mL 100 mMTris-SO₄, pH 7.5. Weigh out 16.3 mg NaN₃, add to the aqueous solution,and swirl to dissolve. To start the reaction, dispense 150 mL ofsubstrate buffer to each well. Seal the deep well reaction plate at 180°C. for 3 sec, then react 3 hrs at room temperature with shaking.

Sample preparation is done as follows. After 3 hrs, add 0.5 mL of MTBEto each well. Seal the plate, and shake at 850 rpm for 10 min at roomtemperature. Centrifuge the plate at 2,000 rpm (3220×g) for 2 min toseparate the phases. Transfer 150 μL of the organic phase from each wellto a shallow well plate (Costar 3365). Seal the plate (Heat-sealer setat 170° C. for 2 sec).

Analyze by normal phase HPLC using the following conditions: 10 μLinjection onto a Daicel Chiralcel AD-H column (0.46 cm×250 mm) equippedwith AD-H guard column. Mobile Phase: 95:5 v/v Heptane/Ethanol;Flow-rate: 1 mL min⁻¹ for 14 min runtime; Column Temperature: 25° C.;detection wavelength: 215 nm. HPLC Retention times: (R)-styrene oxide:3.3 min; (S)-styrene oxide: 3.5 min; (R)-2-azido-1-phenylethanol: 8.0min; (R)-1-azido-2-phenylethanol: 8.6 min; (S)-1-azido-2-phenylethanol:9.3 min; (S)-2-azido-1-phenylethanol: 11.0 min.

The enantioselectivity of the enzyme towards the product of ring openingat the beta position can be calculated from the percent conversion andthe percent enantiomeric excess of the product using the formula ofChen, or be easily visualized by plotting % c vs. % e.e. Engineered HHDHpolypeptides exhibiting altered or improved enantioselectivity incatalyzing the ring opening reaction are listed below in Table 4.

TABLE 4 SEQ ID Residue Differences (relative to NO: SEQ ID NO: 244)Selectivity % e.e. Product 40 W139S; S ++ 116 N113S; W139T; N176S; R +++120 F243L; S + 122 W139M; F243S; S + 124 N176S; F243L; R + 130 E85V;F243S; S ++++ 132 D99N; W139M; S ++++ 136 W139S; F243S; S +++ 140 K121R;N176T; F243S; S + 142 N176T; R ++ 144 W139S; N176S; F243S; S − 146W139S; N176S; R +++ 148 W139M; N176T; R +++ 154 W139M; N176S; F243L; R+++ 156 W139M; N176S; H201R; K204R; R +++ 158 S146P; N176S; F243S; S +++162 W139T; N176D; F243S; S ++ 164 W139M; N176D; R − 166 W139T; S180T;F243S; S ++ 172 W139M; N176D; F243S; R + 244 N/A R + 268 W139M; N176S; R+++ 270 W139S; N176T; R ++ 272 G25C; W139M; N176T; R +++ 274 D80V;W139S; S + 276 L62V; W139M; E197G; S ++ 278 E33G; W139T; N176S; R +++282 T3S; W139M; N176S; R ++++ 284 W139T; N176S; R +++ 286 W139M; S ++++288 E95G; W139M; F243S; S + 290 E85D; W139M; I168V; S ++++ 292 W139T;H201R; S ++ 294 V112A; W139S; S ++ −: 0 to <20% e.e. of product +: 20 to<40% e.e. of product ++: 40 to <60% e.e. of product +++: 60 to <80% e.e.of product ++++: 80 to 100% e.e. of product

Example 3 Engineered HHDH Polypeptides Capable of IncreasedRegioselectivity in Catalyzing the Epoxide Ring Opening of Styrene Oxidewith Azide

A deep well plate containing engineered HHDH enzymes in each well isthawed at room temperature for ˜30 min and centrifuged at 4,000 rpm(3220×g) for 5 min at 4° C. to spin down any condensation.

Prepare a 200 mM styrene oxide stock solution in DMSO by dissolving 33mg/mL. To prepare a 30 mL volume of aqueous substrate buffer, add 1250μL of the 200 mM styrene oxide stock solution to 28.75 mL 100 mMTris-SO₄, pH 7.5. Weigh 16.3 mg NaN₃, add to the aqueous solution, andswirl to mix. To start the reaction, dispense 150 μL of substrate bufferinto each well of the deep well plate. Seal the deep well reaction plateat 180° C. for 3 sec, then react 3 hrs at room temperature with shaking.

Sample preparation for HPLC analysis is done as follows. After 3 hrs,add 0.5 mL of MTBE to each well. Seal the plate, and shake at 850 rpmfor 10 min at room temperature. Centrifuge the plate at 2,000 rpm(3220×g) for 2 min to separate the phases. Transfer 150 μL of theorganic phase from each well to a shallow well plate (Costar 3365). Sealthe plate with heat-sealer set at 170° C. for 2 sec.

Samples are analyzed by normal phase HPLC using the followingconditions: 10 μL injection onto a Daicel Chiralcel AD-H column (0.46cm×250 mm) equipped with AD-H guard column. Mobile Phase: 95:5 v/vHeptane/Ethanol; Flow-rate: 1 mL min-1 for 14 min runtime; ColumnTemperature: 25° C.; detection wavelength: 215 nm. Retention times:(R)-Styrene oxide: 3.3 min; (S)-Styrene oxide: 3.5 min;(R)-2-azido-1-phenylethanol: 8.0 min; (R)-1-azido-2-phenylethanol: 8.6min; (S)-1-azido-2-phenylethanol: 9.3 min; (S)-2-azido-1-phenylethanol:11.0 min.

The regioselectivity of the enzyme towards the alpha or beta positioncan be calculated from the percent conversion to beta product(1-azido-2-phenylethanol) and the percent conversion to alpha product(2-azido-1-phenylethanol). Engineered HHDH polypeptides exhibitingincreased alpha or increased beta regioselectivity in catalyzing thering opening reaction are listed below in Table 5.

TABLE 5 More alpha More beta (relative (relative SEQ ID ResidueDifferences to SEQ to SEQ NO: (relative to SEQ ID NO: 244) ID NO: 244)ID NO: 244) 40 W139S; ++++ 116 N113S; W139T; N176S; ++ 120 F243L; + 122W139M; F243S; + 124 N176S; F243L; + 130 E85V; F243S; + 132 D99N; W139M;++++++ 136 W139S; F243S; + 140 K121R; N176T; F243S; + 142 N176T; + 144W139S; N176S; F243S; + 146 W139S; N176S; ++ 148 W139M; N176T; + 154W139M; N176S; F243L; +++ 156 W139M; N176S; H201R; K204R; ++ 158 S146P;N176S; F243S; + 162 W139T; N176D; F243S; ++ 164 W139M; N176D; +++ 166W139T; S180T; F243S; ++ 172 W139M; N176D; F243S; + 268 W139M; N176S; +268 W139M; N176S; ++ 270 W139S; N176T; ++ 272 G25C; W139M; N176T; ++ 274D80V; W139S; +++ 276 L62V; W139M; E197G; ++++ 280 W139T; N176D; ++ 282T3S; W139M; N176S; ++ 284 W139T; N176S; ++ 286 W139M; +++++++ 290 E85D;W139M; I168V; +++++ 294 V112A; W139S; +++ +: 1 to <2 fold more ++: 2 to<3 fold more +++: 3 to <4 fold more ++++: 4 to <5 fold more +++++: 5 to<6 fold more ++++++: 6 to 7 fold more +++++++: more than 7 fold more

Example 4 Engineered HHDH Polypeptides Having Improved Refractoriness toHalide Inhibition in Catalyzing the Ring Closure Reaction ConvertingPNSHH to PNSO

Assuming normal Michaelis Menten competitive inhibitive enzyme kinetics,from the ratio of initial reaction rate in the absence and presence ofhalide, it is possible to determine the degree of inhibition by halideformula below. V_(obs) is the reaction rate in the presence of halide,V₀ is the uninhibited reaction rate, [I] is the halide concentration(100 mM in this example) and K_(i) is the apparent inhibition constant.

$\begin{matrix}{V_{obs} = {V_{0}*\frac{1}{1 + \frac{\lbrack l\rbrack}{K_{i}}}}} & {{Formula}\mspace{14mu} 1}\end{matrix}$

Determination of uninhibited reaction rate: To make the reactionmixture, mix 1 mL of PNSHH/DMSO stock solution (6.15 mg/mL PNSHH inDMSO) with 99 mL of 100 mM Tris-SO₄ buffer of pH 7.5 by manual shakingvigorously. Add 190 μL of the above solution and 10 μL of crudeengineered HHDH enzyme lysate into UV assay plates using a Multimek orany other 96 channel liquid handler. The final concentration of PNSHH is250 μM. Use a UV/Vis microtiter plate reader for kinetic study.Settings: Wavelength: 310 nm; reaction time 5 mins.

Determination of inhibited reaction rate: To make the reaction mixture,mix 1 mL of PNSHH/DMSO stock solution (6.15 mg/ml PNSHH in DMSO) with 99mL of 100 mM Tris-Cl buffer of pH 7.5 by manual shaking vigorously. Add190 μL of the above solution and 10 μL of crude engineered HHDH enzymelysate into UV assay plates using a Multimek or any other 96 channelliquid handler. The final concentration of PNSHH is 250 μM. Use a UV/Vismicrotiter plate reader for kinetic study. Settings: wavelength: 310 nm,reaction time 5 mins

Engineered HHDH polypeptides exhibiting increased refractoriness tohalide inhibition (e.g., decreased inhibition by halide as shown byincreased apparent K_(i)) in catalyzing the ring closure reactionconverting PNSHH (compound (VI)) to PNSO (compound (VII)) are listedbelow in Table 6.

TABLE 6 K_(i) Fold Improved SEQ ID Residue Differences (relative to SEQID NO: (relative to SEQ ID NO: 244) NO: 244) 28 F12G ++ 90 N167H, L178V++++ 94 Y187S ++ 114 F12Y ++ 118 W139M, N176T, F223L, F243L + 148 W139M,N176T + 172 W139M, N176D, F243S +++ 254 L178V ++ 256 A114V, L178C ++++260 W139S, W249V +++ 268 W139M, N176S + +: 1 to <2 fold higher K_(i) ++:2 to <5 fold higher K_(i) +++: 5 to <10 fold higher K_(i) ++++: 10 to 20fold higher K_(i)

Example 5 Engineered HHDH Polypeptides Capable of Improved Activity inCatalyzing the Non-Terminal Epoxide Ring Closure Reaction of2-Chloro-Cyclohexanol to Cyclohexene Oxide

Thaw a deep well plate containing engineered HHDH cell lysate at roomtemperature for ˜30 min and centrifuge the thawed plate at 4,000 rpm(3220×g) for 5 min at 4° C. to spin down any condensation.

A reaction mixture containing 50 mM 2-chloro-cyclohexanol in 100 mMTris-SO₄, pH 7.5 was prepared. To start the reaction, dispense 150 μL ofsubstrate buffer to each well containing 50 μL of crude engineered HHDHcell lysate. Seal the deep well reaction plate at 180° C. for 3 sec,then react 3 hrs at room temperature with shaking.

Sample preparation for achiral GC analysis is done as follows. After 3hrs, add 1 mL of ethyl acetate to each well. Seal the plate, and shakeat 850 rpm for 10 min at room temperature. Centrifuge the plate at 2,000rpm (3220×g) for 2 min to separate the phases. Transfer 150 μL of theorganic phase from each well to a shallow well plate (Costar 3365). Sealthe plate (Heat-sealer set at 170° C. for 2 sec).

Analysis with achiral GC can be done using a standard Agilent HP5column, 90° C., 10-15 psi, at a 100:1 split ratio, runtime 5 min.Epoxide (2.4 min) and haloalcohol (4.3 min) are easily separated.

Engineered HHDH polypeptides capable of improved activity in catalyzingthe ring closure reaction of 2-chloro-cyclohexanol to cyclohexene oxideare listed below in Table 7.

TABLE 7 Activity Fold Residue differences Improvement SEQ ID (relativeto SEQ ID (relative to SEQ ID NO: NO: 244) NO: 244) 62 A134T; ++ 220V84I; ++ 228 A134V; + 298 V84F; + 302 V84L; +++ 304 V84M; + +: 1 to <2fold more active ++: 2 to 3 fold more active +++: >3 fold more active

Example 6 Engineered HHDH Polypeptides Capable of Improved Activity inthe Non-Terminal Epoxide Ring Opening of Cis-2,3-Epoxybutane with Azideand Trans-2,3-Epoxybutane

Thaw a deep well plate containing engineered HHDH cell lysate at roomtemperature for ˜30 min and centrifuge the thawed plate at 4,000 rpm(3220×g) for 5 min at 4° C. to spin down any condensation.

A mixture of cis or trans epoxybutane in buffer and azide was preparedand dispensed in each well the deep well plate. Final concentrationsepoxybutanes used: 20 mM cis-2,3-epoxy butane (or trans-2,3-epoxybutane) with 20 mM sodium azide. This mixture was reacted for 2 hrs atroom temperature and extracted with MTBE (0.5 mL) containing 5 mMmesitylene as internal standard. GC analysis was done on a standardAgilent HP-1 column, 80° C., 13 psi, injector 250° C. at a 100:1 splitratio, run time 3.5 min. The epoxide is not retained on the column, butthe retention times for the other analytes are: azidoalcohol fromcis-2,3-epoxybutane: 2.24 min⁻¹; azidoalcohol fromtrans-2,3-epoxybutane: 2.33 min⁻¹; mesitylene standard: 3.32 min⁻¹.

Without having the analytically pure product standards, the foldimprovement of conversion needed to be calculated as the rate of productpeak formation relative to the rate of peak formation by parent.

Engineered HHDH polypeptides capable of at least 1.1-fold improvedactivity in the azidolysis of cis-2,3-epoxybutane relative to the HHDHpolypeptide of SEQ ID NO:244 are listed below in Table 8.

TABLE 8 SEQ ID Residue differences Activity Fold Improvement NO:(relative to SEQ ID NO: 244) (relative to SEQ ID NO: 244) 30 F12M; + 64N176H; ++ 66 I63V; N176F; + 68 N176M; ++++ 96 Y187I; ++ 112 D182S; + 178A82F; + 182 A82L; + 186 A82Y; + 210 W86F; + 212 R20C; W86H; G137W; +++214 A134C; + 220 V84I; ++ 226 A134L; +++ 228 A134V; +++ 238 L142M; + 242L142C; + 246 F12M; T67I; + +: 1.1 to <1.5 fold improved ++: 1.5 to <2fold improved +++: 2 to 2.5 fold improved ++++: >2.5 fold improved

Engineered HHDH polypeptides capable of at least 1.1-fold improvedactivity in the azidolysis of trans-2,3-epoxybutane relative to the HHDHpolypeptide of SEQ ID NO:244 are listed below in Table 9.

TABLE 9 SEQ ID Residue differences Activity Fold Improvement NO:(relative to SEQ ID NO: 244) (relative to SEQ ID NO: 244) 30 F12M; + 58P175C; + 64 N176H; + 68 N176M; + 82 A82T; + 96 Y187I; ++ 178 A82F; ++180 A82G; + 182 A82L; + 184 A82S; + 186 A82Y; ++ 202 A82I; + 204 A82K; +206 A82M; ++ 210 W86F; ++ 212 R20C; W86H; G137W; +++ 214 A134C; ++ 216A82N; + 218 A82W; ++ 220 V84I; ++ 226 A134L; +++ 228 A134V; +++++ 230L142G; + 232 A134F; + 234 A134M; + 236 L142I; + 238 L142M; ++ 242 L142C;++ 254 L178V; ++ 258 Y186F; + 296 V84C; + 300 I81L; + +: 1 to <1.5 foldimproved ++: 1.5 to <3 fold improved +++: 3 to <5 fold improved ++++: 5to 10 fold improved +++++: >10 fold improved

Engineered HHDH polypeptides capable of at least 1.1-fold improvedactivity in the azidolysis of both cis- and trans-2,3-epoxybutanerelative to the HHDH polypeptide of SEQ ID NO:244 are listed below inTable 10.

TABLE 10 SEQ Residue differences ID (relative to Activity Fold ActivityFold NO: SEQ ID NO: 244) Improvement cis Improvement trans 30 F12M; + +64 N176H; ++ + 68 N176M; ++ + 96 Y187I; ++ ++ 178 A82F; + ++ 182A82L; + + 186 A82Y; + ++ 210 W86F; + ++ 212 R20C; W86H; G137W; ++ +++214 A134C; + ++ 220 V84I; ++ ++ 226 A134L; ++ +++ 228 A134V; ++ +++++238 L142M; + ++ 242 L142C; + ++ +: 1 to <1.5 fold improved ++: 1.5 to <3fold improved +++: 3 to <5 fold improved ++++: 5 to 10 fold improved+++++: >10 fold improved

Example 7 Engineered HHDH Polypeptides Capable of Catalyzing the RingOpening of an α,α-Disubstituted Styrene Oxide with Formate Nucleophilewith Improved Enantioselectivity

This example illustrates the ability of engineered HHDH polypeptides tocatalyze the ring opening reaction of an α,α-disubstituted styrene oxideof compound of formula (X) using formate as nucleophile, as shown belowin Scheme 12.

A deep well plate containing engineered HHDH polypeptides is thawed atroom temperature for ˜30 min and centrifuged at 4,000 rpm (3220×g) for 5min at 4° C. to spin down any condensation.

A 40 g/L substrate stock solution of the α,α-disubstituted styrene oxideof compound (X) is prepared in DMSO. A 240 mM sodium formate solution isprepared in 100 mM phosphate at pH 6.5. Start the enzyme reaction bydispensing 250 μL of formate buffer and 15 μL of epoxide substrate stocksolution in DMSO to each well of the plate containing the engineeredHHDH polypeptides to give a final concentration in the screening of 2g/L (11.8 mM) epoxide substrate of compound (X). The mixture is allowedto react for 18 hrs at 20° C. at which time it is analyzed by chiralchromatography.

In all samples, even negative control, peaks were detected representingepoxide, diol and two different carbonate esters indicating ring openingof the epoxide with subsequent hydrolysis of the labile formate esterproduct. However, since there was a relatively high rate of chemicalring opening by formate, it was impossible to distinguish betweenregioselectivity (i.e., alpha attack vs. beta attack) andtransesterification of the resulting hydroxy ester. Consequently, thisassay was only capable of identifying those HHDH variants having arelatively high degree of enantioselectivity in the enzymatic ringopening.

As shown in Table 11 below, enantiomeric enrichment in the remainingepoxide demonstrates that enantioselective ring opening of compound (X)using formate as nucleophile is catalyzed by the listed engineered HHDHpolypeptides. Furthermore, the stereoselectivity varies depending on thevariant HHDH used. Based on these results, at least the followingpositions and residue differences affect enantioselectivity of thisreaction: X81E, M, or W; X82C, or S; X84I; X1341; X142G, or N; andX176D.

TABLE 11 SEQ ID Residue Differences % e.e. of remaining NO: (relative toSEQ ID NO: 244) epoxide 4 WT HheC + W249F 12 176 A82C −9 184 A82S −2 194I81E −32 196 I81M −9 198 I81W −22 220 V84I −11 222 A134I 18 230 L142G −5240 L142N −3

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

1. An engineered halohydrin dehalogenase (HDDH) comprising an amino acidsequence at least 90% identical to SEQ ID NO: 244 and at least one aminoacid residue difference at position 139 (X139) as compared to SEQ ID NO:244, wherein said amino acid residue difference at X139 of SEQ ID NO:244 is selected from amino acid residue G, I, L, M, P, S, T or V.
 2. Theengineered HHDH of claim 1, wherein the amino acid sequence has at leastone residue difference as compared to SEQ ID NO:244 selected from: X12is M, or S; X34 is G; X51 is P; X81 is E, F, M, W, or Y; X82 is C, G, K,L, M, N, or W; X83 is C or E; X84 is D; X85 is V; X86 is A or H; X110 isS, X131 is A; X134 is C, F, I, K, L, M, or V; X142 is C, M or N; X146 isP; X167 is H; X175 is C, G, I, L, M, N, or V; X176 is F, M, Q, or T;X177 is L, S or T; X178 is T; X182 is S; X186 is H; X187 is G, I, or S;X201 is R; X232 is S, X242 is A; X243 is C or L; X245 is S; X246 is S;X247 is N; X248 is V; X249 is A, G, H, I, or S; X252 is C; and X254 isN.
 3. The engineered HHDH of claim 1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 24, 32, 34, 36, 38, 40,42, 100, 102, 104, 106, 108, 116, 118, 120, 122, 126, 128, 132, 134,136, 138, 144, 146, 148, 150, 154, 156, 162, 164, 166, 168, 170, 172,260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286,288, 290, 292, and
 294. 4. The engineered HHDH of claim 1 which iscapable of converting a racemic styrene oxide substrate to an S-azidoalcohol product with an enantiomeric excess of at least 20% product andthe amino acid sequence has one or more residue differences or groups ofresidue differences as compared to SEQ ID NO: 244 selected from: X62 isV, X139 is M and X197 is G; X80 is V and X139 is S; X85 is D, X139 is Mand X 168 is V; X85 is V and X243 is S; X95 is G, X139 is M and X243 isS; X99 is N and X139 is M; X112 is A and X139 is S; X121 is R, X176 is Tand X243 is S; X139 is S or M; X139 is T, X176 is D, and X243 is S; X139is T, X180 is T, and X243 is S; X139 is T and X201 is R; X139 is S andX243 is S; X139 is M and X243 is S; X146 is P, X176 is S, and X243 is S;and X243 is L.
 5. The engineered HHDH of claim 4, wherein theenantiomeric excess is at least 60% and the amino acid sequence has oneor more residue differences or groups of residue differences as comparedto SEQ ID NO: 244 selected from: X85 is D, X139 is M and X 168 is V; X85is V and X243 is S; X99 is N and X139 is M; X139 is M; X139 is S andX243 is S; and X243 is L.
 6. The engineered HHDH of any one of claim 1,wherein the HHDH is capable of producing R-azido alcohol from racemicstyrene oxide with at least about 40% enantiomeric excess and the aminoacid sequence has one or more residue differences or groups of residuedifferences as compared to SEQ ID NO: 244 selected from: X25 is C, X139is M, and X176 is T; X33 is G, X139 is T, and X176 is S; X113 is S, X139is T, and X176 is S; X139 is M, and X176 is S; X139 is M, and X176 is T;X139 is S, and X176 is S; X139 is T, and X176 is S; X139 is S, and X176is T; X139 is M, X176 is S, X201 is R, and X204 is R; X139 is M, X176 isS, and X243 is L; and N176T.
 7. The engineered HHDH of claim 6, whereinthe HHDH is capable of producing R-azido alcohol from racemic styreneoxide with at least about 60% enantiomeric excess and the HHDH aminoacid sequence one or more residue differences or groups of residuedifferences as compared to SEQ ID NO: 244 selected from: X25 is C, X139is M, and X176 is T; X33 is G, X139 is T, and X176 is S; X113 is S, X139is T, and X176 is S; X139 is M, and X176 is S; X139 is M, and X176 is T;X139 is S, and X176 is S; X139 is T, and X176 is S; X139 is M, X176 isS, X201 is R, and X204 is R; and X139 is M, X176 is S, and X243 is L. 8.The engineered HHDH of claim 1, wherein the HHDH has at least 3-foldincreased α-regioselectivity as compared to the polypeptide of SEQ IDNO: 244 in the conversion of styrene oxide to the corresponding alcoholproduct and has one or more residue differences or groups of residuedifferences as compared to SEQ ID NO: 244 selected from: X62 is V, X139is M, and X197 is G; X80 is V, and X139 is S; X85 is D, X139 is M, andX168 is V; X99 is N, and X139 is M; X112 is A, and X139 is S; X139 is M;and X139 is S.
 9. The engineered HHDH of claim 1, wherein the HHDH hasat least 2-fold increased β-regioselectivity as compared to thepolypeptide of SEQ ID NO: 244 in the conversion of styrene oxide to thecorresponding alcohol product and has one or more residue differences orgroups of residue differences as compared to SEQ ID NO: 244 selectedfrom: X3 is S, X139 is M, and X176 is S; X25 is C, X139 is M, and X176is T; X113 is S, X139 is T, X176 is S; X139 is S, X176 is S; X139 is M,X176 is S, and X243 is L: X139 is M, X176 is S, X201 is R, and X204 isR; X139 is T, X176 is D, and X243 is S; X139 is M, and X176 is D; X139is T, X180 is T, and X243 is S; X139 is M, and X176 is S; X139 is S, andX176 is T; X139 is T, and X176 is D; and X139 is T, and X176 is S. 10.The engineered HHDH of claim 1, wherein the HHDH has at least 5-foldincreased K_(i) for halide ion as compared to a reference HHDHpolypeptide of SEQ ID NO: 4 and one or more residue differences orgroups of residue differences as compared to SEQ ID NO: 244 selectedfrom: X12 is Y or G; X114 is V, and X178 is C; X139 is M and X176 is S;X139 is M and X176 is T X139 is M, X176is D, and X243 is S; X139S andX249V; X139 is M, X176 is T, X223 is L, and X243 is L; X167 is H, andX178 is V; X178 is V; and X187 is S.
 11. The engineered HHDH of claim 1,wherein the HHDH has an increased activity for converting an epoxidesubstrate of formula (I) to an alcohol product of formula (II) ascompared to a reference polypeptide of SEQ ID NO: 4 or 244, wherein theepoxide substrate is cis- or trans-2,3-epoxybutane.
 12. The engineeredHHDH of claim 1, wherein the HHDH has at least 1.5 fold greater activityin the conversion of 2-chloro-cyclohexanol to cyclohexene oxide ascompared to a reference polypeptide of SEQ ID NO: 244 amino acidsequence has one or more residue differences as compared to SEQ ID NO:244 selected from: X84 is L, I, M, or F; X134 is T or V; and X134 is T,X142 is L, and X245 is V.
 13. The engineered HHDH of claim 1 which iscapable of catalyzing a ring closure reaction convertingpara-nitro-styrene-hydroxy-halide (PNSHH) to para-nitro-styrene oxide(PNSO) with an enantiomeric excess corresponding to an E-value of atleast 20 and has one or more residue differences or groups of residuedifferences as compared to SEQ ID NO: 244 selected from: X114 is V andX178 is C; X175 is G, L, M, or V; or X175 is L, and X222 is A.
 14. Theengineered HHDH of claim 13, wherein the epoxide product in enantiomericexcess is S-para-nitro-styrene oxide (PNSO) and the amino acid sequenceof the engineered HHDH has one or more residue differences or groups ofresidue differences as compared to SEQ ID NO: 244 selected from: X139 isT; X139 is S and X249 is V; X139 is T and X249 is H; or X139 is M, X223is V, and X249 is P.
 15. A method of converting an epoxide of formula(I) to an alcohol of formula (II),

wherein, R¹ is a substituted or unsubstituted alkyl, cylcoalkyl,heteocycloalkyl, aryl, or heteroaryl; R² is H, or substituted orunsubstituted alkyl, cylcoalkyl, heteocycloalkyl, aryl, or heteroaryl;or wherein R¹ and R² forms a ring; said method comprising contacting theepoxide of formula (I) with the engineered HHDH of claim 1 in thepresence of a nucleophile (Nuc⁻) and under reaction conditions suitablefor converting the epoxide to the alcohol product.
 16. The method ofclaim 15, wherein R¹ is a substituted or unsubstituted aryl group, orsubstituted or unsubstituted phenyl group.
 17. The method of claim 15,wherein the epoxide of formula (I) is a non-terminal epoxide.
 18. Themethod of claim 15, wherein the epoxide is selected from the groupconsisting of: styrene oxide; para-nitrostyrene oxide; benzylethyleneoxide; 1,2-epoxybutane; and 1,2 epoxyhexane.
 19. The method of claim 15,wherein the nucleophile is selected from: Br⁻, Cl⁻, I⁻, NO₂ ⁻, N₃ ⁻,CN⁻, OCN⁻, SCN⁻, or formate.